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Insect Models of Innate Immunity
Publications
2009
Kondo J.
[Exploring the "motion" = "function" of the ribosomal A-site molecular switch] Journal Article
In: Tanpakushitsu Kakusan Koso, vol. 54, no. 11, pp. 1356-62, 2009, (0039-9450 (Print) 0039-9450 (Linking) Journal Article Review).
BibTeX | Tags: *Binding, *RNA/genetics, Agents/adverse, Anti-Bacterial, Bacteria/drug, Biosynthesis/genetics, Crystallography, Disorders/genetics, effects, effects/pharmacology, Hearing, Humans, Mutation, Protein, Ribosomes/chemistry/*genetics/*physiology, RNA, Sites, Transfer, Untranslated, WESTHOF, X-Ray
@article{,
title = {[Exploring the "motion" = "function" of the ribosomal A-site molecular switch]},
author = { J. Kondo},
year = {2009},
date = {2009-01-01},
journal = {Tanpakushitsu Kakusan Koso},
volume = {54},
number = {11},
pages = {1356-62},
note = {0039-9450 (Print)
0039-9450 (Linking)
Journal Article
Review},
keywords = {*Binding, *RNA/genetics, Agents/adverse, Anti-Bacterial, Bacteria/drug, Biosynthesis/genetics, Crystallography, Disorders/genetics, effects, effects/pharmacology, Hearing, Humans, Mutation, Protein, Ribosomes/chemistry/*genetics/*physiology, RNA, Sites, Transfer, Untranslated, WESTHOF, X-Ray},
pubstate = {published},
tppubtype = {article}
}
Messmer M, Putz J, Suzuki T, Sauter C, Sissler M, Florentz C
Tertiary network in mammalian mitochondrial tRNAAsp revealed by solution probing and phylogeny Journal Article
In: Nucleic Acids Res, vol. 37, no. 20, pp. 6881-6895, 2009, ISBN: 19767615, (1362-4962 (Electronic) 0305-1048 (Linking) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Asp/*chemistry/*metabolism Transcription, Base Sequence Databases, FLORENTZ, FRUGIER, Genetic, Nucleic Acid Humans Molecular Sequence Data Nucleic Acid Conformation Phylogeny RNA/*chemistry/*metabolism RNA, SISSLER, Transfer, Unité ARN
@article{,
title = {Tertiary network in mammalian mitochondrial tRNAAsp revealed by solution probing and phylogeny},
author = {M Messmer and J Putz and T Suzuki and C Sauter and M Sissler and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19767615},
isbn = {19767615},
year = {2009},
date = {2009-01-01},
journal = {Nucleic Acids Res},
volume = {37},
number = {20},
pages = {6881-6895},
abstract = {Primary and secondary structures of mammalian mitochondrial (mt) tRNAs are divergent from canonical tRNA structures due to highly skewed nucleotide content and large size variability of D- and T-loops. The nonconservation of nucleotides involved in the expected network of tertiary interactions calls into question the rules governing a functional L-shaped three-dimensional (3D) structure. Here, we report the solution structure of human mt-tRNA(Asp) in its native post-transcriptionally modified form and as an in vitro transcript. Probing performed with nuclease S1, ribonuclease V1, dimethylsulfate, diethylpyrocarbonate and lead, revealed several secondary structures for the in vitro transcribed mt-tRNA(Asp) including predominantly the cloverleaf. On the contrary, the native tRNA(Asp) folds into a single cloverleaf structure, highlighting the contribution of the four newly identified post-transcriptional modifications to correct folding. Reactivities of nucleotides and phosphodiester bonds in the native tRNA favor existence of a full set of six classical tertiary interactions between the D-domain and the variable region, forming the core of the 3D structure. Reactivities of D- and T-loop nucleotides support an absence of interactions between these domains. According to multiple sequence alignments and search for conservation of Leontis-Westhof interactions, the tertiary network core building rules apply to all tRNA(Asp) from mammalian mitochondria.},
note = {1362-4962 (Electronic)
0305-1048 (Linking)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Asp/*chemistry/*metabolism Transcription, Base Sequence Databases, FLORENTZ, FRUGIER, Genetic, Nucleic Acid Humans Molecular Sequence Data Nucleic Acid Conformation Phylogeny RNA/*chemistry/*metabolism RNA, SISSLER, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Primary and secondary structures of mammalian mitochondrial (mt) tRNAs are divergent from canonical tRNA structures due to highly skewed nucleotide content and large size variability of D- and T-loops. The nonconservation of nucleotides involved in the expected network of tertiary interactions calls into question the rules governing a functional L-shaped three-dimensional (3D) structure. Here, we report the solution structure of human mt-tRNA(Asp) in its native post-transcriptionally modified form and as an in vitro transcript. Probing performed with nuclease S1, ribonuclease V1, dimethylsulfate, diethylpyrocarbonate and lead, revealed several secondary structures for the in vitro transcribed mt-tRNA(Asp) including predominantly the cloverleaf. On the contrary, the native tRNA(Asp) folds into a single cloverleaf structure, highlighting the contribution of the four newly identified post-transcriptional modifications to correct folding. Reactivities of nucleotides and phosphodiester bonds in the native tRNA favor existence of a full set of six classical tertiary interactions between the D-domain and the variable region, forming the core of the 3D structure. Reactivities of D- and T-loop nucleotides support an absence of interactions between these domains. According to multiple sequence alignments and search for conservation of Leontis-Westhof interactions, the tertiary network core building rules apply to all tRNA(Asp) from mammalian mitochondria.
Messmer M, Gaudry A, Sissler M, Florentz C
Pathology-related mutation A7526G (A9G) helps in the understanding of the 3D structural core of human mitochondrial tRNA(Asp) Journal Article
In: RNA, vol. 15, no. 8, pp. 1462-1468, 2009, ISBN: 19535463, (1469-9001 (Electronic) Letter Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Asp/*chemistry/*genetics/metabolism Transfer RNA Aminoacylation/genetics, Binding Sites/genetics Humans Kinetics Mitochondrial Myopathies/genetics/metabolism/pathology Models, FLORENTZ, Missense Nucleic Acid Conformation RNA/*chemistry/*genetics/metabolism RNA, Molecular Mutation, SISSLER, Transfer, Unité ARN
@article{,
title = {Pathology-related mutation A7526G (A9G) helps in the understanding of the 3D structural core of human mitochondrial tRNA(Asp)},
author = {M Messmer and A Gaudry and M Sissler and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19535463},
isbn = {19535463},
year = {2009},
date = {2009-01-01},
journal = {RNA},
volume = {15},
number = {8},
pages = {1462-1468},
abstract = {More than 130 mutations in human mitochondrial tRNA (mt-tRNA) genes have been correlated with a variety of neurodegenerative and neuromuscular disorders. Their molecular impacts are of mosaic type, affecting various stages of tRNA biogenesis, structure, and/or functions in mt-translation. Knowledge of mammalian mt-tRNA structures per se remains scarce however. Primary and secondary structures deviate from classical tRNAs, while rules for three-dimensional (3D) folding are almost unknown. Here, we take advantage of a myopathy-related mutation A7526G (A9G) in mt-tRNA(Asp) to investigate both the primary molecular impact underlying the pathology and the role of nucleotide 9 in the network of 3D tertiary interactions. Experimental evidence is presented for existence of a 9-12-23 triple in human mt-tRNA(Asp) with a strongly conserved interaction scheme in mammalian mt-tRNAs. Mutation A7526G disrupts the triple interaction and in turn reduces aspartylation efficiency.},
note = {1469-9001 (Electronic)
Letter
Research Support, Non-U.S. Gov't},
keywords = {Asp/*chemistry/*genetics/metabolism Transfer RNA Aminoacylation/genetics, Binding Sites/genetics Humans Kinetics Mitochondrial Myopathies/genetics/metabolism/pathology Models, FLORENTZ, Missense Nucleic Acid Conformation RNA/*chemistry/*genetics/metabolism RNA, Molecular Mutation, SISSLER, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
More than 130 mutations in human mitochondrial tRNA (mt-tRNA) genes have been correlated with a variety of neurodegenerative and neuromuscular disorders. Their molecular impacts are of mosaic type, affecting various stages of tRNA biogenesis, structure, and/or functions in mt-translation. Knowledge of mammalian mt-tRNA structures per se remains scarce however. Primary and secondary structures deviate from classical tRNAs, while rules for three-dimensional (3D) folding are almost unknown. Here, we take advantage of a myopathy-related mutation A7526G (A9G) in mt-tRNA(Asp) to investigate both the primary molecular impact underlying the pathology and the role of nucleotide 9 in the network of 3D tertiary interactions. Experimental evidence is presented for existence of a 9-12-23 triple in human mt-tRNA(Asp) with a strongly conserved interaction scheme in mammalian mt-tRNAs. Mutation A7526G disrupts the triple interaction and in turn reduces aspartylation efficiency.
Giannouli S, Kyritsis A, Malissovas N, Becker H D, Stathopoulos C
On the role of an unusual tRNAGly isoacceptor in Staphylococcus aureus Journal Article
In: Biochimie, vol. 91, no. 3, pp. 344-351, 2009, ISBN: 19014993, (1638-6183 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Bacterial Glycine-tRNA Ligase/genetics/*metabolism Peptide Elongation Factor Tu/metabolism RNA, Gly/*genetics/*metabolism Recombinant Proteins/metabolism Sequence Analysis, KERN Anticodon/metabolism Computational Biology/methods Genes, RNA Staphylococcus aureus/*genetics/*metabolism Transfer RNA Aminoacylation/genetics, Transfer, Unité ARN
@article{,
title = {On the role of an unusual tRNAGly isoacceptor in Staphylococcus aureus},
author = {S Giannouli and A Kyritsis and N Malissovas and H D Becker and C Stathopoulos},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19014993},
isbn = {19014993},
year = {2009},
date = {2009-01-01},
journal = {Biochimie},
volume = {91},
number = {3},
pages = {344-351},
abstract = {In the available Staphylococcus aureus genomes, four different genes have been annotated to encode tRNA(Gly) isoacceptors. Besides their prominent role in protein synthesis, some of them also participate in the formation of pentaglycine bridges during cell wall synthesis. However, until today, it is not known how many and which of them are actually involved in this essential procedure. In the present study we identified, apart from the four annotated tRNA(Gly) genes, a putative pseudogene which encodes and expresses an unusual fifth tRNA(Gly) isoacceptor in S. aureus (as detected via RT-PCR and subsequent direct sequencing analysis). All the in vitro transcribed tRNA(Gly) molecules (including the "pseudogene-encoded" tRNA(Gly)) can be efficiently aminoacylated by the recombinant S. aureus glycyl-tRNA synthetase. Furthermore, bioinformatic analysis suggests that the "pseudo"-tRNA(Gly(UCC)) identified in the present study and two of the annotated isoacceptors bearing the same anticodon carry specific sequence elements that do not favour the strong interaction with EF-Tu that proteinogenic tRNAs would promote. This observation was verified by the differential capacity of Gly-tRNA(Gly) molecules to form ternary complexes with activated S. aureus EF-Tu.GTP. These tRNA(Gly) molecules display high sequence similarities with their S. epidermidis orthologs which also actively participate in cell wall synthesis. Both bioinformatic and biochemical data suggest that in S. aureus these three glycylated tRNA(Gly) isoacceptors that are weak EF-Tu binders, possibly escape protein synthesis and serve as glycine donors for the formation of pentaglycine bridges that are essential for stabilization of the staphylococcal cell wall.},
note = {1638-6183 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Bacterial Glycine-tRNA Ligase/genetics/*metabolism Peptide Elongation Factor Tu/metabolism RNA, Gly/*genetics/*metabolism Recombinant Proteins/metabolism Sequence Analysis, KERN Anticodon/metabolism Computational Biology/methods Genes, RNA Staphylococcus aureus/*genetics/*metabolism Transfer RNA Aminoacylation/genetics, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In the available Staphylococcus aureus genomes, four different genes have been annotated to encode tRNA(Gly) isoacceptors. Besides their prominent role in protein synthesis, some of them also participate in the formation of pentaglycine bridges during cell wall synthesis. However, until today, it is not known how many and which of them are actually involved in this essential procedure. In the present study we identified, apart from the four annotated tRNA(Gly) genes, a putative pseudogene which encodes and expresses an unusual fifth tRNA(Gly) isoacceptor in S. aureus (as detected via RT-PCR and subsequent direct sequencing analysis). All the in vitro transcribed tRNA(Gly) molecules (including the "pseudogene-encoded" tRNA(Gly)) can be efficiently aminoacylated by the recombinant S. aureus glycyl-tRNA synthetase. Furthermore, bioinformatic analysis suggests that the "pseudo"-tRNA(Gly(UCC)) identified in the present study and two of the annotated isoacceptors bearing the same anticodon carry specific sequence elements that do not favour the strong interaction with EF-Tu that proteinogenic tRNAs would promote. This observation was verified by the differential capacity of Gly-tRNA(Gly) molecules to form ternary complexes with activated S. aureus EF-Tu.GTP. These tRNA(Gly) molecules display high sequence similarities with their S. epidermidis orthologs which also actively participate in cell wall synthesis. Both bioinformatic and biochemical data suggest that in S. aureus these three glycylated tRNA(Gly) isoacceptors that are weak EF-Tu binders, possibly escape protein synthesis and serve as glycine donors for the formation of pentaglycine bridges that are essential for stabilization of the staphylococcal cell wall.
Frechin M, Senger B, Brayé M, Kern D, Martin R P, Becker H D
In: Genes Dev, vol. 23, no. 9, pp. 1119-1130, 2009, ISBN: 19417106, (1549-5477 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Amino Acyl/*metabolism RNA-Binding Proteins/*metabolism Saccharomyces cerevisiae/*enzymology/*metabolism Saccharomyces cerevisiae Proteins/*metabolism Transferases/*metabolism, Fungal Glutamate-tRNA Ligase/*metabolism Glutamic Acid/metabolism Mitochondria/*enzymology Protein Binding Protein Transport RNA, KERN Cytoplasm/enzymology Gene Expression Regulation, Transfer, Unité ARN
@article{,
title = {Yeast mitochondrial Gln-tRNA(Gln) is generated by a GatFAB-mediated transamidation pathway involving Arc1p-controlled subcellular sorting of cytosolic GluRS},
author = {M Frechin and B Senger and M Brayé and D Kern and R P Martin and H D Becker},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19417106},
isbn = {19417106},
year = {2009},
date = {2009-01-01},
journal = {Genes Dev},
volume = {23},
number = {9},
pages = {1119-1130},
abstract = {It is impossible to predict which pathway, direct glutaminylation of tRNA(Gln) or tRNA-dependent transamidation of glutamyl-tRNA(Gln), generates mitochondrial glutaminyl-tRNA(Gln) for protein synthesis in a given species. The report that yeast mitochondria import both cytosolic glutaminyl-tRNA synthetase and tRNA(Gln) has challenged the widespread use of the transamidation pathway in organelles. Here we demonstrate that yeast mitochondrial glutaminyl-tRNA(Gln) is in fact generated by a transamidation pathway involving a novel type of trimeric tRNA-dependent amidotransferase (AdT). More surprising is the fact that cytosolic glutamyl-tRNA synthetase ((c)ERS) is imported into mitochondria, where it constitutes the mitochondrial nondiscriminating ERS that generates the mitochondrial mischarged glutamyl-tRNA(Gln) substrate for the AdT. We show that dual localization of (c)ERS is controlled by binding to Arc1p, a tRNA nuclear export cofactor that behaves as a cytosolic anchoring platform for (c)ERS. Expression of Arc1p is down-regulated when yeast cells are switched from fermentation to respiratory metabolism, thus allowing increased import of (c)ERS to satisfy a higher demand of mitochondrial glutaminyl-tRNA(Gln) for mitochondrial protein synthesis. This novel strategy that enables a single protein to be localized in both the cytosol and mitochondria provides a new paradigm for regulation of the dynamic subcellular distribution of proteins between membrane-separated compartments.},
note = {1549-5477 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Amino Acyl/*metabolism RNA-Binding Proteins/*metabolism Saccharomyces cerevisiae/*enzymology/*metabolism Saccharomyces cerevisiae Proteins/*metabolism Transferases/*metabolism, Fungal Glutamate-tRNA Ligase/*metabolism Glutamic Acid/metabolism Mitochondria/*enzymology Protein Binding Protein Transport RNA, KERN Cytoplasm/enzymology Gene Expression Regulation, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
It is impossible to predict which pathway, direct glutaminylation of tRNA(Gln) or tRNA-dependent transamidation of glutamyl-tRNA(Gln), generates mitochondrial glutaminyl-tRNA(Gln) for protein synthesis in a given species. The report that yeast mitochondria import both cytosolic glutaminyl-tRNA synthetase and tRNA(Gln) has challenged the widespread use of the transamidation pathway in organelles. Here we demonstrate that yeast mitochondrial glutaminyl-tRNA(Gln) is in fact generated by a transamidation pathway involving a novel type of trimeric tRNA-dependent amidotransferase (AdT). More surprising is the fact that cytosolic glutamyl-tRNA synthetase ((c)ERS) is imported into mitochondria, where it constitutes the mitochondrial nondiscriminating ERS that generates the mitochondrial mischarged glutamyl-tRNA(Gln) substrate for the AdT. We show that dual localization of (c)ERS is controlled by binding to Arc1p, a tRNA nuclear export cofactor that behaves as a cytosolic anchoring platform for (c)ERS. Expression of Arc1p is down-regulated when yeast cells are switched from fermentation to respiratory metabolism, thus allowing increased import of (c)ERS to satisfy a higher demand of mitochondrial glutaminyl-tRNA(Gln) for mitochondrial protein synthesis. This novel strategy that enables a single protein to be localized in both the cytosol and mitochondria provides a new paradigm for regulation of the dynamic subcellular distribution of proteins between membrane-separated compartments.
Bailly M, Blaise M, Lorber B, Thirup S, Kern D
Isolation, crystallization and preliminary X-ray analysis of the transamidosome, a ribonucleoprotein involved in asparagine formation Journal Article
In: Acta Crystallogr F Struct Biol Commun, vol. 65, no. Pt 6, pp. 577-581, 2009, ISBN: 19478435, (1744-3091 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Amino Acyl/genetics/metabolism RNA, Asn/*biosynthesis Ribonucleoproteins/*isolation & purification/*metabolism Scattering, KERN FLORENTZ Asparagine/*biosynthesis Aspartate-tRNA Ligase/*chemistry/genetics/*metabolism Crystallization Data Collection Escherichia coli/genetics Light RNA, Radiation Statistics as Topic Thermus thermophilus/genetics/metabolism Transfer RNA Aminoacylation X-Ray Diffraction, Transfer, Unité ARN
@article{,
title = {Isolation, crystallization and preliminary X-ray analysis of the transamidosome, a ribonucleoprotein involved in asparagine formation},
author = {M Bailly and M Blaise and B Lorber and S Thirup and D Kern},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19478435},
isbn = {19478435},
year = {2009},
date = {2009-01-01},
journal = {Acta Crystallogr F Struct Biol Commun},
volume = {65},
number = {Pt 6},
pages = {577-581},
abstract = {Thermus thermophilus deprived of asparagine synthetase synthesizes Asn on tRNA(Asn) via a tRNA-dependent pathway involving a nondiscriminating aspartyl-tRNA synthetase that charges Asp onto tRNA(Asn) prior to conversion of the Asp to Asn by GatCAB, a tRNA-dependent amidotransferase. This pathway also constitutes the route of Asn-tRNA(Asn) formation by bacteria and archaea deprived of asparaginyl-tRNA synthetase. The partners involved in tRNA-dependent Asn formation in T. thermophilus assemble into a ternary complex called the transamidosome. This particule produces Asn-tRNA(Asn) in the presence of free Asp, ATP and an amido-group donor. Crystals of the transamidosome from T. thermophilus were obtained in the presence of PEG 4000 in MES-NaOH buffer pH 6.5. They belonged to the primitive monoclinic space group P2(1), with unit-cell parameters a = 115.9},
note = {1744-3091 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Amino Acyl/genetics/metabolism RNA, Asn/*biosynthesis Ribonucleoproteins/*isolation & purification/*metabolism Scattering, KERN FLORENTZ Asparagine/*biosynthesis Aspartate-tRNA Ligase/*chemistry/genetics/*metabolism Crystallization Data Collection Escherichia coli/genetics Light RNA, Radiation Statistics as Topic Thermus thermophilus/genetics/metabolism Transfer RNA Aminoacylation X-Ray Diffraction, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Thermus thermophilus deprived of asparagine synthetase synthesizes Asn on tRNA(Asn) via a tRNA-dependent pathway involving a nondiscriminating aspartyl-tRNA synthetase that charges Asp onto tRNA(Asn) prior to conversion of the Asp to Asn by GatCAB, a tRNA-dependent amidotransferase. This pathway also constitutes the route of Asn-tRNA(Asn) formation by bacteria and archaea deprived of asparaginyl-tRNA synthetase. The partners involved in tRNA-dependent Asn formation in T. thermophilus assemble into a ternary complex called the transamidosome. This particule produces Asn-tRNA(Asn) in the presence of free Asp, ATP and an amido-group donor. Crystals of the transamidosome from T. thermophilus were obtained in the presence of PEG 4000 in MES-NaOH buffer pH 6.5. They belonged to the primitive monoclinic space group P2(1), with unit-cell parameters a = 115.9
2008
Yao P, Zhu B, Jaeger S, Eriani G, Wang E D
Recognition of tRNALeu by Aquifex aeolicus leucyl-tRNA synthetase during the aminoacylation and editing steps Journal Article
In: Nucleic Acids Res, vol. 36, no. 8, pp. 2728-2738, 2008, ISBN: 18367476, (1362-4962 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Amino Acids/chemistry Anticodon/chemistry Bacteria/enzymology/genetics Base Sequence Iodine Leucine-tRNA Ligase/chemistry/*metabolism Molecular Sequence Data Mutagenesis Nucleic Acid Conformation Protein Footprinting RNA, ERIANI, Leu/*chemistry/genetics/metabolism Ribonucleases Substrate Specificity *Transfer RNA Aminoacylation, Transfer, Unité ARN
@article{,
title = {Recognition of tRNALeu by Aquifex aeolicus leucyl-tRNA synthetase during the aminoacylation and editing steps},
author = {P Yao and B Zhu and S Jaeger and G Eriani and E D Wang},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18367476},
isbn = {18367476},
year = {2008},
date = {2008-01-01},
journal = {Nucleic Acids Res},
volume = {36},
number = {8},
pages = {2728-2738},
abstract = {Recognition of tRNA by the cognate aminoacyl-tRNA synthetase during translation is crucial to ensure the correct expression of the genetic code. To understand tRNA(Leu) recognition sets and their evolution, the recognition of tRNA(Leu) by the leucyl-tRNA synthetase (LeuRS) from the primitive hyperthermophilic bacterium Aquifex aeolicus was studied by RNA probing and mutagenesis. The results show that the base A73; the core structure of tRNA formed by the tertiary interactions U8-A14, G18-U55 and G19-C56; and the orientation of the variable arm are critical elements for tRNA(Leu) aminoacylation. Although dispensable for aminoacylation, the anticodon arm carries discrete editing determinants that are required for stabilizing the conformation of the post-transfer editing state and for promoting translocation of the tRNA acceptor arm from the synthetic to the editing site.},
note = {1362-4962 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Amino Acids/chemistry Anticodon/chemistry Bacteria/enzymology/genetics Base Sequence Iodine Leucine-tRNA Ligase/chemistry/*metabolism Molecular Sequence Data Mutagenesis Nucleic Acid Conformation Protein Footprinting RNA, ERIANI, Leu/*chemistry/genetics/metabolism Ribonucleases Substrate Specificity *Transfer RNA Aminoacylation, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Recognition of tRNA by the cognate aminoacyl-tRNA synthetase during translation is crucial to ensure the correct expression of the genetic code. To understand tRNA(Leu) recognition sets and their evolution, the recognition of tRNA(Leu) by the leucyl-tRNA synthetase (LeuRS) from the primitive hyperthermophilic bacterium Aquifex aeolicus was studied by RNA probing and mutagenesis. The results show that the base A73; the core structure of tRNA formed by the tertiary interactions U8-A14, G18-U55 and G19-C56; and the orientation of the variable arm are critical elements for tRNA(Leu) aminoacylation. Although dispensable for aminoacylation, the anticodon arm carries discrete editing determinants that are required for stabilizing the conformation of the post-transfer editing state and for promoting translocation of the tRNA acceptor arm from the synthetic to the editing site.
Simonetti A, Marzi S, Myasnikov A G, Fabbretti A, Yusupov M, Gualerzi C O, Klaholz B P
Structure of the 30S translation initiation complex Journal Article
In: Nature, vol. 455, no. 7211, pp. 416-420, 2008, ISBN: 18758445, (1476-4687 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Cryoelectron Microscopy Crystallography, Messenger/chemistry/genetics/metabolism RNA, Met/chemistry/genetics/metabolism/ultrastructure Ribosome Subunits/chemistry/metabolism/ultrastructure Ribosomes/chemistry/*metabolism/*ultrastructure Thermus thermophilus/*enzymology/genetics/*ultrastructure, Molecular Multiprotein Complexes/*chemistry/genetics/metabolism/*ultrastructure *Peptide Chain Initiation, ROMBY, Transfer, Translational Prokaryotic Initiation Factor-1/chemistry/genetics/metabolism/ultrastructure Prokaryotic Initiation Factor-2/chemistry/genetics/metabolism/ultrastructure Protein Conformation RNA, Unité ARN, X-Ray Guanosine Triphosphate/chemistry/metabolism Models
@article{,
title = {Structure of the 30S translation initiation complex},
author = {A Simonetti and S Marzi and A G Myasnikov and A Fabbretti and M Yusupov and C O Gualerzi and B P Klaholz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18758445},
isbn = {18758445},
year = {2008},
date = {2008-01-01},
journal = {Nature},
volume = {455},
number = {7211},
pages = {416-420},
abstract = {Translation initiation, the rate-limiting step of the universal process of protein synthesis, proceeds through sequential, tightly regulated steps. In bacteria, the correct messenger RNA start site and the reading frame are selected when, with the help of initiation factors IF1, IF2 and IF3, the initiation codon is decoded in the peptidyl site of the 30S ribosomal subunit by the fMet-tRNA(fMet) anticodon. This yields a 30S initiation complex (30SIC) that is an intermediate in the formation of the 70S initiation complex (70SIC) that occurs on joining of the 50S ribosomal subunit to the 30SIC and release of the initiation factors. The localization of IF2 in the 30SIC has proved to be difficult so far using biochemical approaches, but could now be addressed using cryo-electron microscopy and advanced particle separation techniques on the basis of three-dimensional statistical analysis. Here we report the direct visualization of a 30SIC containing mRNA, fMet-tRNA(fMet) and initiation factors IF1 and GTP-bound IF2. We demonstrate that the fMet-tRNA(fMet) is held in a characteristic and precise position and conformation by two interactions that contribute to the formation of a stable complex: one involves the transfer RNA decoding stem which is buried in the 30S peptidyl site, and the other occurs between the carboxy-terminal domain of IF2 and the tRNA acceptor end. The structure provides insights into the mechanism of 70SIC assembly and rationalizes the rapid activation of GTP hydrolysis triggered on 30SIC-50S joining by showing that the GTP-binding domain of IF2 would directly face the GTPase-activated centre of the 50S subunit.},
note = {1476-4687 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Cryoelectron Microscopy Crystallography, Messenger/chemistry/genetics/metabolism RNA, Met/chemistry/genetics/metabolism/ultrastructure Ribosome Subunits/chemistry/metabolism/ultrastructure Ribosomes/chemistry/*metabolism/*ultrastructure Thermus thermophilus/*enzymology/genetics/*ultrastructure, Molecular Multiprotein Complexes/*chemistry/genetics/metabolism/*ultrastructure *Peptide Chain Initiation, ROMBY, Transfer, Translational Prokaryotic Initiation Factor-1/chemistry/genetics/metabolism/ultrastructure Prokaryotic Initiation Factor-2/chemistry/genetics/metabolism/ultrastructure Protein Conformation RNA, Unité ARN, X-Ray Guanosine Triphosphate/chemistry/metabolism Models},
pubstate = {published},
tppubtype = {article}
}
Translation initiation, the rate-limiting step of the universal process of protein synthesis, proceeds through sequential, tightly regulated steps. In bacteria, the correct messenger RNA start site and the reading frame are selected when, with the help of initiation factors IF1, IF2 and IF3, the initiation codon is decoded in the peptidyl site of the 30S ribosomal subunit by the fMet-tRNA(fMet) anticodon. This yields a 30S initiation complex (30SIC) that is an intermediate in the formation of the 70S initiation complex (70SIC) that occurs on joining of the 50S ribosomal subunit to the 30SIC and release of the initiation factors. The localization of IF2 in the 30SIC has proved to be difficult so far using biochemical approaches, but could now be addressed using cryo-electron microscopy and advanced particle separation techniques on the basis of three-dimensional statistical analysis. Here we report the direct visualization of a 30SIC containing mRNA, fMet-tRNA(fMet) and initiation factors IF1 and GTP-bound IF2. We demonstrate that the fMet-tRNA(fMet) is held in a characteristic and precise position and conformation by two interactions that contribute to the formation of a stable complex: one involves the transfer RNA decoding stem which is buried in the 30S peptidyl site, and the other occurs between the carboxy-terminal domain of IF2 and the tRNA acceptor end. The structure provides insights into the mechanism of 70SIC assembly and rationalizes the rapid activation of GTP hydrolysis triggered on 30SIC-50S joining by showing that the GTP-binding domain of IF2 would directly face the GTPase-activated centre of the 50S subunit.
Rederstorff M, Allamand V, Guicheney P, Gartioux C, Richard P, Chaigne D, Krol A, Lescure A
Ex vivo correction of selenoprotein N deficiency in rigid spine muscular dystrophy caused by a mutation in the selenocysteine codon Journal Article
In: Nucleic Acids Res, vol. 36, no. 1, pp. 237-244, 2008, ISBN: 18025044, (1362-4962 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Amino Acid-Specific/*genetics Selenocysteine/metabolism Selenoproteins/biosynthesis/*deficiency/*genetics Transgenes, KROL Codon/chemistry *Codon, LESCURE, Nonsense Fibroblasts/metabolism Hela Cells Humans Muscle Proteins/biosynthesis/*deficiency/*genetics Muscular Atrophy, Spinal/*genetics/metabolism RNA, Transfer, Unité ARN
@article{,
title = {Ex vivo correction of selenoprotein N deficiency in rigid spine muscular dystrophy caused by a mutation in the selenocysteine codon},
author = {M Rederstorff and V Allamand and P Guicheney and C Gartioux and P Richard and D Chaigne and A Krol and A Lescure},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18025044},
isbn = {18025044},
year = {2008},
date = {2008-01-01},
journal = {Nucleic Acids Res},
volume = {36},
number = {1},
pages = {237-244},
abstract = {Premature termination of translation due to nonsense mutations is a frequent cause of inherited diseases. Therefore, many efforts were invested in the development of strategies or compounds to selectively suppress this default. Selenoproteins are interesting candidates considering the idiosyncrasy of the amino acid selenocysteine (Sec) insertion mechanism. Here, we focused our studies on SEPN1, a selenoprotein gene whose mutations entail genetic disorders resulting in different forms of muscular diseases. Selective correction of a nonsense mutation at the Sec codon (UGA to UAA) was undertaken with a corrector tRNA(Sec) that was engineered to harbor a compensatory mutation in the anticodon. We demonstrated that its expression restored synthesis of a full-length selenoprotein N both in HeLa cells and in skin fibroblasts from a patient carrying the mutated Sec codon. Readthrough of the UAA codon was effectively dependent on the Sec insertion machinery, therefore being highly selective for this gene and unlikely to generate off-target effects. In addition, we observed that expression of the corrector tRNA(Sec) stabilized the mutated SEPN1 transcript that was otherwise more subject to degradation. In conclusion, our data provide interesting evidence that premature termination of translation due to nonsense mutations is amenable to correction, in the context of the specialized selenoprotein synthesis mechanism.},
note = {1362-4962 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Amino Acid-Specific/*genetics Selenocysteine/metabolism Selenoproteins/biosynthesis/*deficiency/*genetics Transgenes, KROL Codon/chemistry *Codon, LESCURE, Nonsense Fibroblasts/metabolism Hela Cells Humans Muscle Proteins/biosynthesis/*deficiency/*genetics Muscular Atrophy, Spinal/*genetics/metabolism RNA, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Premature termination of translation due to nonsense mutations is a frequent cause of inherited diseases. Therefore, many efforts were invested in the development of strategies or compounds to selectively suppress this default. Selenoproteins are interesting candidates considering the idiosyncrasy of the amino acid selenocysteine (Sec) insertion mechanism. Here, we focused our studies on SEPN1, a selenoprotein gene whose mutations entail genetic disorders resulting in different forms of muscular diseases. Selective correction of a nonsense mutation at the Sec codon (UGA to UAA) was undertaken with a corrector tRNA(Sec) that was engineered to harbor a compensatory mutation in the anticodon. We demonstrated that its expression restored synthesis of a full-length selenoprotein N both in HeLa cells and in skin fibroblasts from a patient carrying the mutated Sec codon. Readthrough of the UAA codon was effectively dependent on the Sec insertion machinery, therefore being highly selective for this gene and unlikely to generate off-target effects. In addition, we observed that expression of the corrector tRNA(Sec) stabilized the mutated SEPN1 transcript that was otherwise more subject to degradation. In conclusion, our data provide interesting evidence that premature termination of translation due to nonsense mutations is amenable to correction, in the context of the specialized selenoprotein synthesis mechanism.
Pujol C, Bailly M, Kern D, Marechal-Drouard L, Becker H, Duchene A M
Dual-targeted tRNA-dependent amidotransferase ensures both mitochondrial and chloroplastic Gln-tRNAGln synthesis in plants Journal Article
In: Proc Natl Acad Sci U S A, vol. 105, no. 17, pp. 6481-6485, 2008, ISBN: 18441100, (1091-6490 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Amino Acyl/*biosynthesis Solanum tuberosum/*enzymology, KERN Arabidopsis/*enzymology Cell Extracts Chloroplasts/*enzymology Cytosol/enzymology Glutamate-tRNA Ligase/metabolism Glutamine/*biosynthesis Mitochondria/*enzymology Nitrogenous Group Transferases/*metabolism Protein Subunits/metabolism Protein Transport RNA, Transfer, Unité ARN
@article{,
title = {Dual-targeted tRNA-dependent amidotransferase ensures both mitochondrial and chloroplastic Gln-tRNAGln synthesis in plants},
author = {C Pujol and M Bailly and D Kern and L Marechal-Drouard and H Becker and A M Duchene},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18441100},
isbn = {18441100},
year = {2008},
date = {2008-01-01},
journal = {Proc Natl Acad Sci U S A},
volume = {105},
number = {17},
pages = {6481-6485},
abstract = {Aminoacyl-tRNAs are generally formed by direct attachment of an amino acid to tRNAs by aminoacyl-tRNA synthetases, but Gln-tRNA is an exception to this rule. Gln-tRNA(Gln) is formed by this direct pathway in the eukaryotic cytosol and in protists or fungi mitochondria but is formed by an indirect transamidation pathway in most of bacteria, archaea, and chloroplasts. We show here that the formation of Gln-tRNA(Gln) is also achieved by the indirect pathway in plant mitochondria. The mitochondrial-encoded tRNA(Gln), which is the only tRNA(Gln) present in mitochondria, is first charged with glutamate by a nondiscriminating GluRS, then is converted into Gln-tRNA(Gln) by a tRNA-dependent amidotransferase (AdT). The three subunits GatA, GatB, and GatC are imported into mitochondria and assemble into a functional GatCAB AdT. Moreover, the mitochondrial pathway of Gln-tRNA(Gln) formation is shared with chloroplasts as both the GluRS, and the three AdT subunits are dual-imported into mitochondria and chloroplasts.},
note = {1091-6490 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Amino Acyl/*biosynthesis Solanum tuberosum/*enzymology, KERN Arabidopsis/*enzymology Cell Extracts Chloroplasts/*enzymology Cytosol/enzymology Glutamate-tRNA Ligase/metabolism Glutamine/*biosynthesis Mitochondria/*enzymology Nitrogenous Group Transferases/*metabolism Protein Subunits/metabolism Protein Transport RNA, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Aminoacyl-tRNAs are generally formed by direct attachment of an amino acid to tRNAs by aminoacyl-tRNA synthetases, but Gln-tRNA is an exception to this rule. Gln-tRNA(Gln) is formed by this direct pathway in the eukaryotic cytosol and in protists or fungi mitochondria but is formed by an indirect transamidation pathway in most of bacteria, archaea, and chloroplasts. We show here that the formation of Gln-tRNA(Gln) is also achieved by the indirect pathway in plant mitochondria. The mitochondrial-encoded tRNA(Gln), which is the only tRNA(Gln) present in mitochondria, is first charged with glutamate by a nondiscriminating GluRS, then is converted into Gln-tRNA(Gln) by a tRNA-dependent amidotransferase (AdT). The three subunits GatA, GatB, and GatC are imported into mitochondria and assemble into a functional GatCAB AdT. Moreover, the mitochondrial pathway of Gln-tRNA(Gln) formation is shared with chloroplasts as both the GluRS, and the three AdT subunits are dual-imported into mitochondria and chloroplasts.
Bonnefond L, Florentz C, Giege R, Rudinger-Thirion J
Decreased aminoacylation in pathology-related mutants of mitochondrial tRNATyr is associated with structural perturbations in tRNA architecture Journal Article
In: RNA, vol. 14, no. 4, pp. 641-648, 2008, ISBN: 18268021, (1469-9001 (Electronic) In Vitro Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Base Sequence Humans Mitochondrial Diseases/genetics/metabolism Models, FLORENTZ, FRUGIER, Molecular Molecular Sequence Data Nucleic Acid Conformation *Point Mutation RNA/*chemistry/*genetics/metabolism RNA Stability RNA, Transfer, Tyr/*chemistry/*genetics/metabolism *Transfer RNA Aminoacylation, Unité ARN
@article{,
title = {Decreased aminoacylation in pathology-related mutants of mitochondrial tRNATyr is associated with structural perturbations in tRNA architecture},
author = {L Bonnefond and C Florentz and R Giege and J Rudinger-Thirion},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18268021},
isbn = {18268021},
year = {2008},
date = {2008-01-01},
journal = {RNA},
volume = {14},
number = {4},
pages = {641-648},
abstract = {A growing number of human pathologies are ascribed to mutations in mitochondrial tRNA genes. Here, we report biochemical investigations on three mt-tRNA(Tyr) molecules with point substitutions associated with diseases. The mutations occur in the atypical T- and D-loops at positions homologous to those involved in the tertiary interaction network of canonical tRNAs. They do not correspond to tyrosine identity positions and likely do not contact the mitochondrial tyrosyl-tRNA synthetase during the aminoacylation process. The impact of these substitutions on mt-tRNA(Tyr) tyrosylation and structure was investigated using the corresponding tRNA transcripts. In vitro tyrosylation efficiency is decreased 600-fold for mutant A22G (mitochondrial gene mutation T5874C), 40-fold for G15A (C5877T), and is without significant effect on U54C (A5843G). Comparative solution probings with lead and nucleases on mutant and wild-type tRNA(Tyr) molecules reveal a greater sensitivity to single-strand specific probes for mutants G15A and A22G. For both transcripts, the mutation triggers a structural destabilization in the D-loop that propagates toward the anticodon arm and thus hinders efficient tyrosylation. Further probing analysis combined with phylogenetic data support the participation of G15 and A22 in the tertiary network of human mt-tRNA(Tyr) via nonclassical Watson-Crick G15-C48 and G13-A22 pairings. In contrast, the pathogenic effect of the tyrosylable mutant U54C, where structure is only marginally affected, has to be sought at another level of the tRNA(Tyr) life cycle.},
note = {1469-9001 (Electronic)
In Vitro
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Base Sequence Humans Mitochondrial Diseases/genetics/metabolism Models, FLORENTZ, FRUGIER, Molecular Molecular Sequence Data Nucleic Acid Conformation *Point Mutation RNA/*chemistry/*genetics/metabolism RNA Stability RNA, Transfer, Tyr/*chemistry/*genetics/metabolism *Transfer RNA Aminoacylation, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
A growing number of human pathologies are ascribed to mutations in mitochondrial tRNA genes. Here, we report biochemical investigations on three mt-tRNA(Tyr) molecules with point substitutions associated with diseases. The mutations occur in the atypical T- and D-loops at positions homologous to those involved in the tertiary interaction network of canonical tRNAs. They do not correspond to tyrosine identity positions and likely do not contact the mitochondrial tyrosyl-tRNA synthetase during the aminoacylation process. The impact of these substitutions on mt-tRNA(Tyr) tyrosylation and structure was investigated using the corresponding tRNA transcripts. In vitro tyrosylation efficiency is decreased 600-fold for mutant A22G (mitochondrial gene mutation T5874C), 40-fold for G15A (C5877T), and is without significant effect on U54C (A5843G). Comparative solution probings with lead and nucleases on mutant and wild-type tRNA(Tyr) molecules reveal a greater sensitivity to single-strand specific probes for mutants G15A and A22G. For both transcripts, the mutation triggers a structural destabilization in the D-loop that propagates toward the anticodon arm and thus hinders efficient tyrosylation. Further probing analysis combined with phylogenetic data support the participation of G15 and A22 in the tertiary network of human mt-tRNA(Tyr) via nonclassical Watson-Crick G15-C48 and G13-A22 pairings. In contrast, the pathogenic effect of the tyrosylable mutant U54C, where structure is only marginally affected, has to be sought at another level of the tRNA(Tyr) life cycle.
Blaise M, Olieric V, Sauter C, Lorber B, Roy B, Karmakar S, Banerjee R, Becker H D, Kern D
In: J Mol Biol, vol. 381, no. 5, pp. 1224-1237, 2008, ISBN: 18602926, (1089-8638 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Adenosine Triphosphate/metabolism Amino Acid Sequence Amino Acyl-tRNA Synthetases/*chemistry Anticodon/*metabolism Base Sequence Binding Sites Catalysis Conserved Sequence Crystallography, Asp/*chemistry/genetics Regulatory Sequences, FFRUGIER, Molecular Molecular Sequence Data Nucleic Acid Conformation Nucleoside Q/*chemistry Protein Structure, Ribonucleic Acid/*genetics Thermus thermophilus/enzymology, Secondary RNA, Transfer, Unité ARN, X-Ray Escherichia coli/*enzymology Escherichia coli Proteins/*chemistry Glutamic Acid/*chemistry Models
@article{,
title = {Crystal structure of glutamyl-queuosine tRNAAsp synthetase complexed with L-glutamate: structural elements mediating tRNA-independent activation of glutamate and glutamylation of tRNAAsp anticodon},
author = {M Blaise and V Olieric and C Sauter and B Lorber and B Roy and S Karmakar and R Banerjee and H D Becker and D Kern},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18602926},
isbn = {18602926},
year = {2008},
date = {2008-01-01},
journal = {J Mol Biol},
volume = {381},
number = {5},
pages = {1224-1237},
abstract = {Glutamyl-queuosine tRNA(Asp) synthetase (Glu-Q-RS) from Escherichia coli is a paralog of the catalytic core of glutamyl-tRNA synthetase (GluRS) that catalyzes glutamylation of queuosine in the wobble position of tRNA(Asp). Despite important structural similarities, Glu-Q-RS and GluRS diverge strongly by their functional properties. The only feature common to both enzymes consists in the activation of Glu to form Glu-AMP, the intermediate of transfer RNA (tRNA) aminoacylation. However, both enzymes differ by the mechanism of selection of the cognate amino acid and by the mechanism of its activation. Whereas GluRS selects l-Glu and activates it only in the presence of the cognate tRNA(Glu), Glu-Q-RS forms Glu-AMP in the absence of tRNA. Moreover, while GluRS transfers the activated Glu to the 3' accepting end of the cognate tRNA(Glu), Glu-Q-RS transfers the activated Glu to Q34 located in the anticodon loop of the noncognate tRNA(Asp). In order to gain insight into the structural elements leading to distinct mechanisms of amino acid activation, we solved the three-dimensional structure of Glu-Q-RS complexed to Glu and compared it to the structure of the GluRS.Glu complex. Comparison of the catalytic site of Glu-Q-RS with that of GluRS, combined with binding experiments of amino acids, shows that a restricted number of residues determine distinct catalytic properties of amino acid recognition and activation by the two enzymes. Furthermore, to explore the structural basis of the distinct aminoacylation properties of the two enzymes and to understand why Glu-Q-RS glutamylates only tRNA(Asp) among the tRNAs possessing queuosine in position 34, we performed a tRNA mutational analysis to search for the elements of tRNA(Asp) that determine recognition by Glu-Q-RS. The analyses made on tRNA(Asp) and tRNA(Asn) show that the presence of a C in position 38 is crucial for glutamylation of Q34. The results are discussed in the context of the evolution and adaptation of the tRNA glutamylation system.},
note = {1089-8638 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Adenosine Triphosphate/metabolism Amino Acid Sequence Amino Acyl-tRNA Synthetases/*chemistry Anticodon/*metabolism Base Sequence Binding Sites Catalysis Conserved Sequence Crystallography, Asp/*chemistry/genetics Regulatory Sequences, FFRUGIER, Molecular Molecular Sequence Data Nucleic Acid Conformation Nucleoside Q/*chemistry Protein Structure, Ribonucleic Acid/*genetics Thermus thermophilus/enzymology, Secondary RNA, Transfer, Unité ARN, X-Ray Escherichia coli/*enzymology Escherichia coli Proteins/*chemistry Glutamic Acid/*chemistry Models},
pubstate = {published},
tppubtype = {article}
}
Glutamyl-queuosine tRNA(Asp) synthetase (Glu-Q-RS) from Escherichia coli is a paralog of the catalytic core of glutamyl-tRNA synthetase (GluRS) that catalyzes glutamylation of queuosine in the wobble position of tRNA(Asp). Despite important structural similarities, Glu-Q-RS and GluRS diverge strongly by their functional properties. The only feature common to both enzymes consists in the activation of Glu to form Glu-AMP, the intermediate of transfer RNA (tRNA) aminoacylation. However, both enzymes differ by the mechanism of selection of the cognate amino acid and by the mechanism of its activation. Whereas GluRS selects l-Glu and activates it only in the presence of the cognate tRNA(Glu), Glu-Q-RS forms Glu-AMP in the absence of tRNA. Moreover, while GluRS transfers the activated Glu to the 3' accepting end of the cognate tRNA(Glu), Glu-Q-RS transfers the activated Glu to Q34 located in the anticodon loop of the noncognate tRNA(Asp). In order to gain insight into the structural elements leading to distinct mechanisms of amino acid activation, we solved the three-dimensional structure of Glu-Q-RS complexed to Glu and compared it to the structure of the GluRS.Glu complex. Comparison of the catalytic site of Glu-Q-RS with that of GluRS, combined with binding experiments of amino acids, shows that a restricted number of residues determine distinct catalytic properties of amino acid recognition and activation by the two enzymes. Furthermore, to explore the structural basis of the distinct aminoacylation properties of the two enzymes and to understand why Glu-Q-RS glutamylates only tRNA(Asp) among the tRNAs possessing queuosine in position 34, we performed a tRNA mutational analysis to search for the elements of tRNA(Asp) that determine recognition by Glu-Q-RS. The analyses made on tRNA(Asp) and tRNA(Asn) show that the presence of a C in position 38 is crucial for glutamylation of Q34. The results are discussed in the context of the evolution and adaptation of the tRNA glutamylation system.
2007
Roy H, Becker H D, Mazauric M H, Kern D
Structural elements defining elongation factor Tu mediated suppression of codon ambiguity Journal Article
In: Nucleic Acids Res, vol. 35, no. 10, pp. 3420-3430, 2007, ISBN: 17478519, (1362-4962 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Amino Acyl/*chemistry/metabolism RNA, Asn/*chemistry/metabolism RNA, Asp/chemistry/metabolism Thermus thermophilus/genetics *Transfer RNA Aminoacylation, KERN Base Pairing *Codon Escherichia coli Proteins/metabolism Models, Molecular Peptide Elongation Factor Tu/*chemistry/metabolism Protein Binding RNA, Transfer, Unité ARN
@article{,
title = {Structural elements defining elongation factor Tu mediated suppression of codon ambiguity},
author = {H Roy and H D Becker and M H Mazauric and D Kern},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17478519},
isbn = {17478519},
year = {2007},
date = {2007-01-01},
journal = {Nucleic Acids Res},
volume = {35},
number = {10},
pages = {3420-3430},
abstract = {In most prokaryotes Asn-tRNA(Asn) and Gln-tRNA(Gln) are formed by amidation of aspartate and glutamate mischarged onto tRNA(Asn) and tRNA(Gln), respectively. Coexistence in the organism of mischarged Asp-tRNA(Asn) and Glu-tRNA(Gln) and the homologous Asn-tRNA(Asn) and Gln-tRNA(Gln) does not, however, lead to erroneous incorporation of Asp and Glu into proteins, since EF-Tu discriminates the misacylated tRNAs from the correctly charged ones. This property contrasts with the canonical function of EF-Tu, which is to non-specifically bind the homologous aa-tRNAs, as well as heterologous species formed in vitro by aminoacylation of non-cognate tRNAs. In Thermus thermophilus that forms the Asp-tRNA(Asn) intermediate by the indirect pathway of tRNA asparaginylation, EF-Tu must discriminate the mischarged aminoacyl-tRNAs (aa-tRNA). We show that two base pairs in the tRNA T-arm and a single residue in the amino acid binding pocket of EF-Tu promote discrimination of Asp-tRNA(Asn) from Asn-tRNA(Asn) and Asp-tRNA(Asp) by the protein. Our analysis suggests that these structural elements might also contribute to rejection of other mischarged aa-tRNAs formed in vivo that are not involved in peptide elongation. Additionally, these structural features might be involved in maintaining a delicate balance of weak and strong binding affinities between EF-Tu and the amino acid and tRNA moieties of other elongator aa-tRNAs.},
note = {1362-4962 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Amino Acyl/*chemistry/metabolism RNA, Asn/*chemistry/metabolism RNA, Asp/chemistry/metabolism Thermus thermophilus/genetics *Transfer RNA Aminoacylation, KERN Base Pairing *Codon Escherichia coli Proteins/metabolism Models, Molecular Peptide Elongation Factor Tu/*chemistry/metabolism Protein Binding RNA, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In most prokaryotes Asn-tRNA(Asn) and Gln-tRNA(Gln) are formed by amidation of aspartate and glutamate mischarged onto tRNA(Asn) and tRNA(Gln), respectively. Coexistence in the organism of mischarged Asp-tRNA(Asn) and Glu-tRNA(Gln) and the homologous Asn-tRNA(Asn) and Gln-tRNA(Gln) does not, however, lead to erroneous incorporation of Asp and Glu into proteins, since EF-Tu discriminates the misacylated tRNAs from the correctly charged ones. This property contrasts with the canonical function of EF-Tu, which is to non-specifically bind the homologous aa-tRNAs, as well as heterologous species formed in vitro by aminoacylation of non-cognate tRNAs. In Thermus thermophilus that forms the Asp-tRNA(Asn) intermediate by the indirect pathway of tRNA asparaginylation, EF-Tu must discriminate the mischarged aminoacyl-tRNAs (aa-tRNA). We show that two base pairs in the tRNA T-arm and a single residue in the amino acid binding pocket of EF-Tu promote discrimination of Asp-tRNA(Asn) from Asn-tRNA(Asn) and Asp-tRNA(Asp) by the protein. Our analysis suggests that these structural elements might also contribute to rejection of other mischarged aa-tRNAs formed in vivo that are not involved in peptide elongation. Additionally, these structural features might be involved in maintaining a delicate balance of weak and strong binding affinities between EF-Tu and the amino acid and tRNA moieties of other elongator aa-tRNAs.
Henriet S, Sinck L, Bec G, Gorelick R J, Marquet R, Paillart J C
Vif is a RNA chaperone that could temporally regulate RNA dimerization and the early steps of HIV-1 reverse transcription Journal Article
In: Nucleic Acids Res, vol. 35, no. 15, pp. 5141-5153, 2007, ISBN: 17660191, (1362-4962 (Electronic) Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Amino Acyl/metabolism RNA, Capsid Proteins/metabolism DNA, gag/metabolism Gene Products, Human Immunodeficiency Virus, Human Immunodeficiency Virus vif Gene Products, MARQUET, PAILLART, Single-Stranded/biosynthesis Dimerization Gene Products, Transfer, Unité ARN, vif/*metabolism HIV-1/*genetics Molecular Chaperones/*metabolism RNA, Viral/*metabolism *Reverse Transcription Viral Proteins/metabolism gag Gene Products
@article{,
title = {Vif is a RNA chaperone that could temporally regulate RNA dimerization and the early steps of HIV-1 reverse transcription},
author = {S Henriet and L Sinck and G Bec and R J Gorelick and R Marquet and J C Paillart},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17660191},
isbn = {17660191},
year = {2007},
date = {2007-01-01},
journal = {Nucleic Acids Res},
volume = {35},
number = {15},
pages = {5141-5153},
abstract = {HIV-1 Vif (viral infectivity factor) is associated with the assembly complexes and packaged at low level into the viral particles, and is essential for viral replication in non-permissive cells. Viral particles produced in the absence of Vif exhibit structural defects and are defective in the early steps of reverse transcription. Here, we show that Vif is able to anneal primer tRNA(Lys3) to the viral RNA, to decrease pausing of reverse transcriptase during (-) strand strong-stop DNA synthesis, and to promote the first strand transfer. Vif also stimulates formation of loose HIV-1 genomic RNA dimers. These results indicate that Vif is a bona fide RNA chaperone. We next studied the effects of Vif in the presence of HIV-1 NCp, which is a well-established RNA chaperone. Vif inhibits NCp-mediated formation of tight RNA dimers and hybridization of tRNA(Lys3), while it has little effects on NCp-mediated strand transfer and it collaborates with nucleocapsid (NC) to increase RT processivity. Thus, Vif might negatively regulate NC-assisted maturation of the RNA dimer and early steps of reverse transcription in the assembly complexes, but these inhibitory effects would be relieved after viral budding, thanks to the limited packaging of Vif in the virions.},
note = {1362-4962 (Electronic)
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't},
keywords = {Amino Acyl/metabolism RNA, Capsid Proteins/metabolism DNA, gag/metabolism Gene Products, Human Immunodeficiency Virus, Human Immunodeficiency Virus vif Gene Products, MARQUET, PAILLART, Single-Stranded/biosynthesis Dimerization Gene Products, Transfer, Unité ARN, vif/*metabolism HIV-1/*genetics Molecular Chaperones/*metabolism RNA, Viral/*metabolism *Reverse Transcription Viral Proteins/metabolism gag Gene Products},
pubstate = {published},
tppubtype = {article}
}
HIV-1 Vif (viral infectivity factor) is associated with the assembly complexes and packaged at low level into the viral particles, and is essential for viral replication in non-permissive cells. Viral particles produced in the absence of Vif exhibit structural defects and are defective in the early steps of reverse transcription. Here, we show that Vif is able to anneal primer tRNA(Lys3) to the viral RNA, to decrease pausing of reverse transcriptase during (-) strand strong-stop DNA synthesis, and to promote the first strand transfer. Vif also stimulates formation of loose HIV-1 genomic RNA dimers. These results indicate that Vif is a bona fide RNA chaperone. We next studied the effects of Vif in the presence of HIV-1 NCp, which is a well-established RNA chaperone. Vif inhibits NCp-mediated formation of tight RNA dimers and hybridization of tRNA(Lys3), while it has little effects on NCp-mediated strand transfer and it collaborates with nucleocapsid (NC) to increase RT processivity. Thus, Vif might negatively regulate NC-assisted maturation of the RNA dimer and early steps of reverse transcription in the assembly complexes, but these inhibitory effects would be relieved after viral budding, thanks to the limited packaging of Vif in the virions.
Maone E, Stefano M Di, Berardi A, Benelli D, Marzi S, Teana A La, Londei P
Functional analysis of the translation factor aIF2/5B in the thermophilic archaeon Sulfolobus solfataricus Journal Article
In: Mol Microbiol, vol. 65, no. 3, pp. 700-13, 2007, ISBN: 17608795, (0950-382X (Print) 0950-382X (Linking) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Archaeal Hydrolysis Peptide Chain Initiation, Met/metabolism Recombinant Fusion Proteins/metabolism Ribosomes/metabolism Sulfolobus solfataricus/genetics/*metabolism, Molecular Conserved Sequence GTP Phosphohydrolases/metabolism Gene Expression Genes, ROMBY, Secondary RNA, Transfer, Translational Peptide Initiation Factors/chemistry/isolation & purification/*metabolism Protein Binding *Protein Biosynthesis Protein Structure, Unité ARN
@article{,
title = {Functional analysis of the translation factor aIF2/5B in the thermophilic archaeon Sulfolobus solfataricus},
author = {E Maone and M Di Stefano and A Berardi and D Benelli and S Marzi and A La Teana and P Londei},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17608795},
doi = {10.1111/j.1365-2958.2007.05820.x},
isbn = {17608795},
year = {2007},
date = {2007-01-01},
journal = {Mol Microbiol},
volume = {65},
number = {3},
pages = {700-13},
abstract = {The protein IF2/eIF5B is one of the few translation initiation factors shared by all three primary domains of life (bacteria, archaea, eukarya). Despite its phylogenetic conservation, the factor is known to present marked functional divergences in the bacteria and the eukarya. In this work, the function in translation of the archaeal homologue (aIF2/5B) has been analysed in detail for the first time using a variety of in vitro assays. The results revealed that the protein is a ribosome-dependent GTPase which strongly stimulates the binding of initiator tRNA to the ribosomes even in the absence of other factors. In agreement with this finding, aIF2/5B enhances the translation of both leadered and leaderless mRNAs when expressed in a cell-free protein-synthesizing system. Moreover, the degree of functional conservation of the IF2-like factors in the archaeal and bacterial lineages was investigated by analysing the behaviour of 'chimeric' proteins produced by swapping domains between the Sulfolobus solfataricus aIF2/5B factor and the IF2 protein of the thermophilic bacterium Bacillus stearothermophilus. Beside evidencing similarities and differences between the archaeal and bacterial factors, these experiments have provided insight into the common role played by the IF2/5B proteins in all extant cells.},
note = {0950-382X (Print)
0950-382X (Linking)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Archaeal Hydrolysis Peptide Chain Initiation, Met/metabolism Recombinant Fusion Proteins/metabolism Ribosomes/metabolism Sulfolobus solfataricus/genetics/*metabolism, Molecular Conserved Sequence GTP Phosphohydrolases/metabolism Gene Expression Genes, ROMBY, Secondary RNA, Transfer, Translational Peptide Initiation Factors/chemistry/isolation & purification/*metabolism Protein Binding *Protein Biosynthesis Protein Structure, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The protein IF2/eIF5B is one of the few translation initiation factors shared by all three primary domains of life (bacteria, archaea, eukarya). Despite its phylogenetic conservation, the factor is known to present marked functional divergences in the bacteria and the eukarya. In this work, the function in translation of the archaeal homologue (aIF2/5B) has been analysed in detail for the first time using a variety of in vitro assays. The results revealed that the protein is a ribosome-dependent GTPase which strongly stimulates the binding of initiator tRNA to the ribosomes even in the absence of other factors. In agreement with this finding, aIF2/5B enhances the translation of both leadered and leaderless mRNAs when expressed in a cell-free protein-synthesizing system. Moreover, the degree of functional conservation of the IF2-like factors in the archaeal and bacterial lineages was investigated by analysing the behaviour of 'chimeric' proteins produced by swapping domains between the Sulfolobus solfataricus aIF2/5B factor and the IF2 protein of the thermophilic bacterium Bacillus stearothermophilus. Beside evidencing similarities and differences between the archaeal and bacterial factors, these experiments have provided insight into the common role played by the IF2/5B proteins in all extant cells.
2006
Westhof E
The ribosomal decoding site and antibiotics Journal Article
In: Biochimie, vol. 88, no. 8, pp. 931-933, 2006, ISBN: 16876930, (0300-9084 (Print) Editorial).
Links | BibTeX | Tags: Anti-Bacterial Agents/metabolism/*pharmacology Bacteria/*drug effects/genetics/growth & development Binding Sites/drug effects Protein Biosynthesis/drug effects/genetics RNA Amino Acyl/*metabolism, Bacterial/*metabolism RNA, Transfer, Unité ARN, WESTHOF
@article{,
title = {The ribosomal decoding site and antibiotics},
author = {E Westhof},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16876930},
isbn = {16876930},
year = {2006},
date = {2006-01-01},
journal = {Biochimie},
volume = {88},
number = {8},
pages = {931-933},
note = {0300-9084 (Print)
Editorial},
keywords = {Anti-Bacterial Agents/metabolism/*pharmacology Bacteria/*drug effects/genetics/growth & development Binding Sites/drug effects Protein Biosynthesis/drug effects/genetics RNA Amino Acyl/*metabolism, Bacterial/*metabolism RNA, Transfer, Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
Fender A, Sauter C, Messmer M, Putz J, Giege R, Florentz C, Sissler M
Loss of a primordial identity element for a mammalian mitochondrial aminoacylation system Journal Article
In: J Biol Chem, vol. 281, no. 23, pp. 15980-15986, 2006, ISBN: 16597625, (0021-9258 (Print) Journal Article).
Abstract | Links | BibTeX | Tags: Acylation Base Sequence Humans Kinetics Mutagenesis Nucleic Acid Conformation Plasmids RNA | Non-U.S. Gov't, Asp/chemistry/genetics/*metabolism Research Support, FLORENTZ, FRUGIER, Non-U.S. Gov't, SISSLER, Transfer, Unité ARN
@article{,
title = {Loss of a primordial identity element for a mammalian mitochondrial aminoacylation system},
author = {A Fender and C Sauter and M Messmer and J Putz and R Giege and C Florentz and M Sissler},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16597625},
isbn = {16597625},
year = {2006},
date = {2006-01-01},
journal = {J Biol Chem},
volume = {281},
number = {23},
pages = {15980-15986},
abstract = {In mammalian mitochondria the translational machinery is of dual origin with tRNAs encoded by a simplified and rapidly evolving mitochondrial (mt) genome and aminoacyl-tRNA synthetases (aaRS) coded by the nuclear genome, and imported. Mt-tRNAs are atypical with biased sequences, size variations in loops and stems, and absence of residues forming classical tertiary interactions, whereas synthetases appear typical. This raises questions about identity elements in mt-tRNAs and adaptation of their cognate mt-aaRSs. We have explored here the human mt-aspartate system in which a prokaryotic-type AspRS, highly similar to the Escherichia coli enzyme, recognizes a bizarre tRNA(Asp). Analysis of human mt-tRNA(Asp) transcripts confirms the identity role of the GUC anticodon as in other aspartylation systems but reveals the non-involvement of position 73. This position is otherwise known as the site of a universally conserved major aspartate identity element, G73, also known as a primordial identity signal. In mt-tRNA(Asp), position 73 can be occupied by any of the four nucleotides without affecting aspartylation. Sequence alignments of various AspRSs allowed placing Gly-269 at a position occupied by Asp-220, the residue contacting G73 in the crystallographic structure of E. coli AspRS-tRNA(Asp) complex. Replacing this glycine by an aspartate renders human mt-AspRS more discriminative to G73. Restriction in the aspartylation identity set, driven by a rapid mutagenic rate of the mt-genome, suggests a reverse evolution of the mt-tRNA(Asp) identity elements in regard to its bacterial ancestor.},
note = {0021-9258 (Print)
Journal Article},
keywords = {Acylation Base Sequence Humans Kinetics Mutagenesis Nucleic Acid Conformation Plasmids RNA | Non-U.S. Gov't, Asp/chemistry/genetics/*metabolism Research Support, FLORENTZ, FRUGIER, Non-U.S. Gov't, SISSLER, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In mammalian mitochondria the translational machinery is of dual origin with tRNAs encoded by a simplified and rapidly evolving mitochondrial (mt) genome and aminoacyl-tRNA synthetases (aaRS) coded by the nuclear genome, and imported. Mt-tRNAs are atypical with biased sequences, size variations in loops and stems, and absence of residues forming classical tertiary interactions, whereas synthetases appear typical. This raises questions about identity elements in mt-tRNAs and adaptation of their cognate mt-aaRSs. We have explored here the human mt-aspartate system in which a prokaryotic-type AspRS, highly similar to the Escherichia coli enzyme, recognizes a bizarre tRNA(Asp). Analysis of human mt-tRNA(Asp) transcripts confirms the identity role of the GUC anticodon as in other aspartylation systems but reveals the non-involvement of position 73. This position is otherwise known as the site of a universally conserved major aspartate identity element, G73, also known as a primordial identity signal. In mt-tRNA(Asp), position 73 can be occupied by any of the four nucleotides without affecting aspartylation. Sequence alignments of various AspRSs allowed placing Gly-269 at a position occupied by Asp-220, the residue contacting G73 in the crystallographic structure of E. coli AspRS-tRNA(Asp) complex. Replacing this glycine by an aspartate renders human mt-AspRS more discriminative to G73. Restriction in the aspartylation identity set, driven by a rapid mutagenic rate of the mt-genome, suggests a reverse evolution of the mt-tRNA(Asp) identity elements in regard to its bacterial ancestor.
2005
Jenner L, Romby P, Rees B, Schulze-Briese C, Springer M, Ehresmann C, Ehresmann B, Moras D, Yusupova G, Yusupov M
Translational operator of mRNA on the ribosome: how repressor proteins exclude ribosome binding Journal Article
In: Science, vol. 308, no. 5718, pp. 120-123, 2005, ISBN: 15802605, (1095-9203 Journal Article).
Abstract | Links | BibTeX | Tags: 16S/chemistry/metabolism RNA, Bacterial/*chemistry/metabolism RNA, Messenger/*chemistry/metabolism RNA, Met/chemistry/metabolism *Regulatory Sequences, Molecular Nucleic Acid Conformation *Protein Biosynthesis RNA, Non-U.S. Gov't Ribosomal Proteins/metabolism Ribosomes/*metabolism Thermus thermophilus/genetics/*metabolism Threonine-tRNA Ligase/genetics/metabolism, Ribonucleic Acid Repressor Proteins/*metabolism Research Support, Ribosomal, ROMBY, ROMBY Bacterial Proteins/metabolism Base Pairing Binding Sites Crystallization Crystallography, Transfer, Unité ARN, X-Ray Fourier Analysis Models
@article{,
title = {Translational operator of mRNA on the ribosome: how repressor proteins exclude ribosome binding},
author = {L Jenner and P Romby and B Rees and C Schulze-Briese and M Springer and C Ehresmann and B Ehresmann and D Moras and G Yusupova and M Yusupov},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15802605},
isbn = {15802605},
year = {2005},
date = {2005-01-01},
journal = {Science},
volume = {308},
number = {5718},
pages = {120-123},
abstract = {The ribosome of Thermus thermophilus was cocrystallized with initiator transfer RNA (tRNA) and a structured messenger RNA (mRNA) carrying a translational operator. The path of the mRNA was defined at 5.5 angstroms resolution by comparing it with either the crystal structure of the same ribosomal complex lacking mRNA or with an unstructured mRNA. A precise ribosomal environment positions the operator stem-loop structure perpendicular to the surface of the ribosome on the platform of the 30S subunit. The binding of the operator and of the initiator tRNA occurs on the ribosome with an unoccupied tRNA exit site, which is expected for an initiation complex. The positioning of the regulatory domain of the operator relative to the ribosome elucidates the molecular mechanism by which the bound repressor switches off translation. Our data suggest a general way in which mRNA control elements must be placed on the ribosome to perform their regulatory task.},
note = {1095-9203
Journal Article},
keywords = {16S/chemistry/metabolism RNA, Bacterial/*chemistry/metabolism RNA, Messenger/*chemistry/metabolism RNA, Met/chemistry/metabolism *Regulatory Sequences, Molecular Nucleic Acid Conformation *Protein Biosynthesis RNA, Non-U.S. Gov't Ribosomal Proteins/metabolism Ribosomes/*metabolism Thermus thermophilus/genetics/*metabolism Threonine-tRNA Ligase/genetics/metabolism, Ribonucleic Acid Repressor Proteins/*metabolism Research Support, Ribosomal, ROMBY, ROMBY Bacterial Proteins/metabolism Base Pairing Binding Sites Crystallization Crystallography, Transfer, Unité ARN, X-Ray Fourier Analysis Models},
pubstate = {published},
tppubtype = {article}
}
The ribosome of Thermus thermophilus was cocrystallized with initiator transfer RNA (tRNA) and a structured messenger RNA (mRNA) carrying a translational operator. The path of the mRNA was defined at 5.5 angstroms resolution by comparing it with either the crystal structure of the same ribosomal complex lacking mRNA or with an unstructured mRNA. A precise ribosomal environment positions the operator stem-loop structure perpendicular to the surface of the ribosome on the platform of the 30S subunit. The binding of the operator and of the initiator tRNA occurs on the ribosome with an unoccupied tRNA exit site, which is expected for an initiation complex. The positioning of the regulatory domain of the operator relative to the ribosome elucidates the molecular mechanism by which the bound repressor switches off translation. Our data suggest a general way in which mRNA control elements must be placed on the ribosome to perform their regulatory task.
Graindorge J S, Senger B, Tritch D, Simos G, Fasiolo F
Role of Arc1p in the modulation of yeast glutamyl-tRNA synthetase activity Journal Article
In: Biochemistry, vol. 44, no. 4, pp. 1344-1352, 2005, ISBN: 15667228, (0006-2960 Journal Article).
Abstract | Links | BibTeX | Tags: FASIOLO Adenosine Triphosphate/chemistry/metabolism Amino Acid Sequence Aminoacylation Base Sequence Diphosphates/chemistry/metabolism Enzyme Activation Gene Expression Regulation, Fungal Glutamate-tRNA Ligase/isolation & purification/*metabolism Kinetics Molecular Sequence Data Peptide Fragments/chemistry/metabolism Protein Binding Protein Structure, Fungal/genetics/metabolism RNA, Genetic, Glu/genetics/metabolism RNA-Binding Proteins/*chemistry/isolation & purification/metabolism Research Support, Non-U.S. Gov't Saccharomyces cerevisiae/enzymology/genetics/metabolism Saccharomyces cerevisiae Proteins/*chemistry/isolation & purification/metabolism Transcription, Tertiary RNA, Transfer, Unité ARN
@article{,
title = {Role of Arc1p in the modulation of yeast glutamyl-tRNA synthetase activity},
author = {J S Graindorge and B Senger and D Tritch and G Simos and F Fasiolo},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15667228},
isbn = {15667228},
year = {2005},
date = {2005-01-01},
journal = {Biochemistry},
volume = {44},
number = {4},
pages = {1344-1352},
abstract = {Yeast methionyl-tRNA synthetase (MetRS) and glutamyl-tRNA synthetase (GluRS) possess N-terminal extensions that bind the cofactor Arc1p in trans. The strength of GluRS-Arc1p interaction is high enough to allow copurification of the two macromolecules in a 1:1 ratio, in contrast to MetRS. Deletion analysis from the C-terminal end of the GluRS appendix combined with previous N-terminal deletions of GluRS allows restriction of the Arc1p binding site to the 110-170 amino acid region of GluRS. This region has been shown to correspond to a novel protein-protein interaction domain present in both GluRS and Arc1p but not in MetRS [Galani, K., Grosshans, H., Deinert, K., Hurt, E. C., and Simos, G. (2001) EMBO J. 20, 6889-6898]. The GluRS apoenzyme fails to show significant kinetics of tRNA aminoacylation and charges unfractionated yeast tRNA at a level 10-fold reduced compared to Arc1p-bound GluRS. The K(m) values for tRNA(Glu) measured in the ATP-PP(i) exchange were similar for the two forms of GluRS, whereas k(cat) is increased 2-fold in the presence of Arc1p. Band-shift analysis revealed a 100-fold increase in tRNA binding affinity when Arc1p is bound to GluRS. This increase requires the RNA binding properties of the full-length Arc1p since Arc1p N domain leaves the K(d) of GluRS for tRNA unchanged. Transcripts of yeast tRNA(Glu) were poor substrates for measuring tRNA aminoacylation and could not be used to clarify whether Arc1p has a specific effect on the tRNA charging reaction.},
note = {0006-2960
Journal Article},
keywords = {FASIOLO Adenosine Triphosphate/chemistry/metabolism Amino Acid Sequence Aminoacylation Base Sequence Diphosphates/chemistry/metabolism Enzyme Activation Gene Expression Regulation, Fungal Glutamate-tRNA Ligase/isolation & purification/*metabolism Kinetics Molecular Sequence Data Peptide Fragments/chemistry/metabolism Protein Binding Protein Structure, Fungal/genetics/metabolism RNA, Genetic, Glu/genetics/metabolism RNA-Binding Proteins/*chemistry/isolation & purification/metabolism Research Support, Non-U.S. Gov't Saccharomyces cerevisiae/enzymology/genetics/metabolism Saccharomyces cerevisiae Proteins/*chemistry/isolation & purification/metabolism Transcription, Tertiary RNA, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Yeast methionyl-tRNA synthetase (MetRS) and glutamyl-tRNA synthetase (GluRS) possess N-terminal extensions that bind the cofactor Arc1p in trans. The strength of GluRS-Arc1p interaction is high enough to allow copurification of the two macromolecules in a 1:1 ratio, in contrast to MetRS. Deletion analysis from the C-terminal end of the GluRS appendix combined with previous N-terminal deletions of GluRS allows restriction of the Arc1p binding site to the 110-170 amino acid region of GluRS. This region has been shown to correspond to a novel protein-protein interaction domain present in both GluRS and Arc1p but not in MetRS [Galani, K., Grosshans, H., Deinert, K., Hurt, E. C., and Simos, G. (2001) EMBO J. 20, 6889-6898]. The GluRS apoenzyme fails to show significant kinetics of tRNA aminoacylation and charges unfractionated yeast tRNA at a level 10-fold reduced compared to Arc1p-bound GluRS. The K(m) values for tRNA(Glu) measured in the ATP-PP(i) exchange were similar for the two forms of GluRS, whereas k(cat) is increased 2-fold in the presence of Arc1p. Band-shift analysis revealed a 100-fold increase in tRNA binding affinity when Arc1p is bound to GluRS. This increase requires the RNA binding properties of the full-length Arc1p since Arc1p N domain leaves the K(d) of GluRS for tRNA unchanged. Transcripts of yeast tRNA(Glu) were poor substrates for measuring tRNA aminoacylation and could not be used to clarify whether Arc1p has a specific effect on the tRNA charging reaction.
Bonnefond L, Frugier M, Giege R, Rudinger-Thirion J
Human mitochondrial TyrRS disobeys the tyrosine identity rules Journal Article
In: RNA, vol. 11, no. 5, pp. 558-562, 2005, ISBN: 15840810, (1355-8382 Journal Article).
Abstract | Links | BibTeX | Tags: Amino Acid Sequence Base Sequence Catalytic Domain Humans Mitochondria/*enzymology Molecular Sequence Data RNA, FRUGIER, Non-U.S. Gov't Substrate Specificity Tyrosine/genetics/*metabolism Tyrosine-tRNA Ligase/chemistry/*metabolism, Transfer, Tyr/genetics/*metabolism Research Support, Unité ARN
@article{,
title = {Human mitochondrial TyrRS disobeys the tyrosine identity rules},
author = {L Bonnefond and M Frugier and R Giege and J Rudinger-Thirion},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15840810},
isbn = {15840810},
year = {2005},
date = {2005-01-01},
journal = {RNA},
volume = {11},
number = {5},
pages = {558-562},
abstract = {Human tyrosyl-tRNA synthetase from mitochondria (mt-TyrRS) presents dual sequence features characteristic of eubacterial and archaeal TyrRSs, especially in the region containing amino acids recognizing the N1-N72 tyrosine identity pair. This would imply that human mt-TyrRS has lost the capacity to discriminate between the G1-C72 pair typical of eubacterial and mitochondrial tRNATyr and the reverse pair C1-G72 present in archaeal and eukaryal tRNATyr. This expectation was verified by a functional analysis of wild-type or mutated tRNATyr molecules, showing that mt-TyrRS aminoacylates with similar catalytic efficiency its cognate tRNATyr with G1-C72 and its mutated version with C1-G72. This provides the first example of a TyrRS lacking specificity toward N1-N72 and thus of a TyrRS disobeying the identity rules. Sequence comparisons of mt-TyrRSs across phylogeny suggest that the functional behavior of the human mt-TyrRS is conserved among all vertebrate mt-TyrRSs.},
note = {1355-8382
Journal Article},
keywords = {Amino Acid Sequence Base Sequence Catalytic Domain Humans Mitochondria/*enzymology Molecular Sequence Data RNA, FRUGIER, Non-U.S. Gov't Substrate Specificity Tyrosine/genetics/*metabolism Tyrosine-tRNA Ligase/chemistry/*metabolism, Transfer, Tyr/genetics/*metabolism Research Support, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Human tyrosyl-tRNA synthetase from mitochondria (mt-TyrRS) presents dual sequence features characteristic of eubacterial and archaeal TyrRSs, especially in the region containing amino acids recognizing the N1-N72 tyrosine identity pair. This would imply that human mt-TyrRS has lost the capacity to discriminate between the G1-C72 pair typical of eubacterial and mitochondrial tRNATyr and the reverse pair C1-G72 present in archaeal and eukaryal tRNATyr. This expectation was verified by a functional analysis of wild-type or mutated tRNATyr molecules, showing that mt-TyrRS aminoacylates with similar catalytic efficiency its cognate tRNATyr with G1-C72 and its mutated version with C1-G72. This provides the first example of a TyrRS lacking specificity toward N1-N72 and thus of a TyrRS disobeying the identity rules. Sequence comparisons of mt-TyrRSs across phylogeny suggest that the functional behavior of the human mt-TyrRS is conserved among all vertebrate mt-TyrRSs.
2004
Costa A., de Barros J. P. Pais, Keith G., Baranowski W., Desgres J.
Determination of queuosine derivatives by reverse-phase liquid chromatography for the hypomodification study of Q-bearing tRNAs from various mammal liver cells Journal Article
In: J Chromatogr B Analyt Technol Biomed Life Sci, vol. 801, no. 2, pp. 237-47, 2004, (1570-0232 Journal Article).
Abstract | BibTeX | Tags: *Chromatography, &, Acyl/chemistry, Amino, Animals, Asn/chemistry, Cells, Chickens, Cultured, derivatives/*analysis, Experimental, Gov't, Hepatocytes/chemistry, high, KEITH, liquid, Liver, Liver/*chemistry, Neoplasms, Non-U.S., Nucleoside, Pressure, purification, Q/*analogs, Rats, RNA, Support, Transfer, Transfer/*chemistry/isolation, tumor
@article{,
title = {Determination of queuosine derivatives by reverse-phase liquid chromatography for the hypomodification study of Q-bearing tRNAs from various mammal liver cells},
author = { A. Costa and J. P. Pais de Barros and G. Keith and W. Baranowski and J. Desgres},
year = {2004},
date = {2004-01-01},
journal = {J Chromatogr B Analyt Technol Biomed Life Sci},
volume = {801},
number = {2},
pages = {237-47},
abstract = {Three queuosine derivatives (Q-derivatives) have been found at position 34 of four mammalian so-called Q-tRNAs: queuosine (Q) in tRNA(Asn) and tRNA(His), mannosyl-queuosine (manQ) in tRNA(Asp), and galactosyl-queuosine (galQ) in tRNA(Tyr). An analytical procedure based on the combined means of purified tRNA isolation from liver cells and ribonucleoside analysis by reverse-phase high performance liquid chromatography coupled with real-time UV-spectrometry (RPLC-UV) was developed for the quantitative analysis of the three Q-derivatives present in total tRNA from liver tissues and liver cell cultures. Using this analytical procedure, the rates of Q-tRNA modification were studied in total tRNAs from various mammalian hepatic cells. Our results show that the four Q-tRNAs are fully modified in liver tissues from adult mammals, regardless of the mammal species. However, a lack in the Q-modification level was observed in Q-tRNAs from newborn rat liver, as well in Q-tRNAs from normal rat liver cell cultures growing in a low queuine content medium, and from a rat hepatoma cell line. It is noteworthy that in all cases of Q-tRNA hypomodification, our analytical procedure showed that tRNA(Asp) is always the least affected by the hypomodification. The biological significance of this phenomenon is discussed.},
note = {1570-0232
Journal Article},
keywords = {*Chromatography, &, Acyl/chemistry, Amino, Animals, Asn/chemistry, Cells, Chickens, Cultured, derivatives/*analysis, Experimental, Gov't, Hepatocytes/chemistry, high, KEITH, liquid, Liver, Liver/*chemistry, Neoplasms, Non-U.S., Nucleoside, Pressure, purification, Q/*analogs, Rats, RNA, Support, Transfer, Transfer/*chemistry/isolation, tumor},
pubstate = {published},
tppubtype = {article}
}
Three queuosine derivatives (Q-derivatives) have been found at position 34 of four mammalian so-called Q-tRNAs: queuosine (Q) in tRNA(Asn) and tRNA(His), mannosyl-queuosine (manQ) in tRNA(Asp), and galactosyl-queuosine (galQ) in tRNA(Tyr). An analytical procedure based on the combined means of purified tRNA isolation from liver cells and ribonucleoside analysis by reverse-phase high performance liquid chromatography coupled with real-time UV-spectrometry (RPLC-UV) was developed for the quantitative analysis of the three Q-derivatives present in total tRNA from liver tissues and liver cell cultures. Using this analytical procedure, the rates of Q-tRNA modification were studied in total tRNAs from various mammalian hepatic cells. Our results show that the four Q-tRNAs are fully modified in liver tissues from adult mammals, regardless of the mammal species. However, a lack in the Q-modification level was observed in Q-tRNAs from newborn rat liver, as well in Q-tRNAs from normal rat liver cell cultures growing in a low queuine content medium, and from a rat hepatoma cell line. It is noteworthy that in all cases of Q-tRNA hypomodification, our analytical procedure showed that tRNA(Asp) is always the least affected by the hypomodification. The biological significance of this phenomenon is discussed.
Martineau Y., Bec C. Le, Monbrun L., Allo V., Chiu I. M., Danos O., Moine H., Prats H., Prats A. C.
Internal ribosome entry site structural motifs conserved among mammalian fibroblast growth factor 1 alternatively spliced mRNAs Journal Article
In: Mol Cell Biol, vol. 24, no. 17, pp. 7622-35, 2004, (0270-7306 Journal Article).
Abstract | BibTeX | Tags: (Genetics), *5', *Alternative, *Nucleic, *Promoter, 1/*genetics, Acid, Alignment, Animals, Base, Cell, Conformation, Data, EHRESMANN, Factor, Fibroblast, Gene, Genes, Genetic, Gov't, Growth, Human, Line, Messenger/chemistry/*genetics/metabolism, Mice, Molecular, Muscle, Mutagenesis, Non-U.S., Regions, Ribosomes/*metabolism, RNA, Sequence, Site-Directed, Skeletal/cytology/physiology, Splicing, Structural/genetics, Support, Techniques, Transfer, Untranslated, Vectors
@article{,
title = {Internal ribosome entry site structural motifs conserved among mammalian fibroblast growth factor 1 alternatively spliced mRNAs},
author = { Y. Martineau and C. Le Bec and L. Monbrun and V. Allo and I. M. Chiu and O. Danos and H. Moine and H. Prats and A. C. Prats},
year = {2004},
date = {2004-01-01},
journal = {Mol Cell Biol},
volume = {24},
number = {17},
pages = {7622-35},
abstract = {Fibroblast growth factor 1 (FGF-1) is a powerful angiogenic factor whose gene structure contains four promoters, giving rise to a process of alternative splicing resulting in four mRNAs with alternative 5' untranslated regions (5' UTRs). Here we have identified, by using double luciferase bicistronic vectors, the presence of internal ribosome entry sites (IRESs) in the human FGF-1 5' UTRs, particularly in leaders A and C, with distinct activities in mammalian cells. DNA electrotransfer in mouse muscle revealed that the IRES present in the FGF-1 leader A has a high activity in vivo. We have developed a new regulatable TET OFF bicistronic system, which allowed us to rule out the possibility of any cryptic promoter in the FGF-1 leaders. FGF-1 IRESs A and C, which were mapped in fragments of 118 and 103 nucleotides, respectively, are flexible in regard to the position of the initiation codon, making them interesting from a biotechnological point of view. Furthermore, we show that FGF-1 IRESs A of murine and human origins show similar IRES activity profiles. Enzymatic and chemical probing of the FGF-1 IRES A RNA revealed a structural domain conserved among mammals at both the nucleotide sequence and RNA structure levels. The functional role of this structural motif has been demonstrated by point mutagenesis, including compensatory mutations. These data favor an important role of IRESs in the control of FGF-1 expression and provide a new IRES structural motif that could help IRES prediction in 5' UTR databases.},
note = {0270-7306
Journal Article},
keywords = {(Genetics), *5', *Alternative, *Nucleic, *Promoter, 1/*genetics, Acid, Alignment, Animals, Base, Cell, Conformation, Data, EHRESMANN, Factor, Fibroblast, Gene, Genes, Genetic, Gov't, Growth, Human, Line, Messenger/chemistry/*genetics/metabolism, Mice, Molecular, Muscle, Mutagenesis, Non-U.S., Regions, Ribosomes/*metabolism, RNA, Sequence, Site-Directed, Skeletal/cytology/physiology, Splicing, Structural/genetics, Support, Techniques, Transfer, Untranslated, Vectors},
pubstate = {published},
tppubtype = {article}
}
Fibroblast growth factor 1 (FGF-1) is a powerful angiogenic factor whose gene structure contains four promoters, giving rise to a process of alternative splicing resulting in four mRNAs with alternative 5' untranslated regions (5' UTRs). Here we have identified, by using double luciferase bicistronic vectors, the presence of internal ribosome entry sites (IRESs) in the human FGF-1 5' UTRs, particularly in leaders A and C, with distinct activities in mammalian cells. DNA electrotransfer in mouse muscle revealed that the IRES present in the FGF-1 leader A has a high activity in vivo. We have developed a new regulatable TET OFF bicistronic system, which allowed us to rule out the possibility of any cryptic promoter in the FGF-1 leaders. FGF-1 IRESs A and C, which were mapped in fragments of 118 and 103 nucleotides, respectively, are flexible in regard to the position of the initiation codon, making them interesting from a biotechnological point of view. Furthermore, we show that FGF-1 IRESs A of murine and human origins show similar IRES activity profiles. Enzymatic and chemical probing of the FGF-1 IRES A RNA revealed a structural domain conserved among mammals at both the nucleotide sequence and RNA structure levels. The functional role of this structural motif has been demonstrated by point mutagenesis, including compensatory mutations. These data favor an important role of IRESs in the control of FGF-1 expression and provide a new IRES structural motif that could help IRES prediction in 5' UTR databases.
Zheng Y G, Wei H, Ling C, Martin F, Eriani G, Wang E D
In: Nucleic Acids Res, vol. 32, no. 11, pp. 3294-3303, 2004, ISBN: 15208367, (1362-4962 Evaluation Studies Journal Article).
Abstract | Links | BibTeX | Tags: Amino Acid Sequence Binding Sites Gram-Negative Bacteria/*enzymology Kinetics Leucine-tRNA Ligase/*chemistry/genetics/*metabolism Molecular Sequence Data Mutation Protein Structure, ERIANI, Leu/*metabolism Saccharomyces cerevisiae/genetics *Two-Hybrid System Techniques, Tertiary Protein Subunits/chemistry RNA, Transfer, Unité ARN
@article{,
title = {Two distinct domains of the beta subunit of Aquifex aeolicus leucyl-tRNA synthetase are involved in tRNA binding as revealed by a three-hybrid selection},
author = {Y G Zheng and H Wei and C Ling and F Martin and G Eriani and E D Wang},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15208367},
isbn = {15208367},
year = {2004},
date = {2004-01-01},
journal = {Nucleic Acids Res},
volume = {32},
number = {11},
pages = {3294-3303},
abstract = {The Aquifex aeolicus alphabeta-LeuRS is the only known heterodimeric class Ia aminoacyl-tRNA synthetase. In this study, we investigated the function of the beta subunit which is believed to bind tRNA(Leu). A yeast three-hybrid system was constructed on the basis of the interaction of the beta subunit with its cognate tRNA(Leu). Then, seven mutated beta subunits exhibiting impaired tRNA binding capacities were selected out from a randomly mutated library. Two mutations were identified in the class Ia-helix-bundle-domain, which might interact with the D-hairpin of the tRNA analogous to other class Ia tRNA:synthetases complexes. The five other mutations were found in the LeuRS-specific C-terminal domain of which the folding is still unknown. tRNA affinity measurements and kinetic analyses performed on the isolated beta subunits and on the co-expressed alphabeta-heterodimers showed for all the mutants an effect in tRNA affinity in the ground state. In addition, an effect on the transition state of the aminoacylation reaction was observed for a 21-residues deletion mutant of the C-terminal end. These results show that the genetic approach of the three hybrid system is widely applicable and is a powerful tool for the investigation of tRNA:synthetase interactions.},
note = {1362-4962
Evaluation Studies
Journal Article},
keywords = {Amino Acid Sequence Binding Sites Gram-Negative Bacteria/*enzymology Kinetics Leucine-tRNA Ligase/*chemistry/genetics/*metabolism Molecular Sequence Data Mutation Protein Structure, ERIANI, Leu/*metabolism Saccharomyces cerevisiae/genetics *Two-Hybrid System Techniques, Tertiary Protein Subunits/chemistry RNA, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The Aquifex aeolicus alphabeta-LeuRS is the only known heterodimeric class Ia aminoacyl-tRNA synthetase. In this study, we investigated the function of the beta subunit which is believed to bind tRNA(Leu). A yeast three-hybrid system was constructed on the basis of the interaction of the beta subunit with its cognate tRNA(Leu). Then, seven mutated beta subunits exhibiting impaired tRNA binding capacities were selected out from a randomly mutated library. Two mutations were identified in the class Ia-helix-bundle-domain, which might interact with the D-hairpin of the tRNA analogous to other class Ia tRNA:synthetases complexes. The five other mutations were found in the LeuRS-specific C-terminal domain of which the folding is still unknown. tRNA affinity measurements and kinetic analyses performed on the isolated beta subunits and on the co-expressed alphabeta-heterodimers showed for all the mutants an effect in tRNA affinity in the ground state. In addition, an effect on the transition state of the aminoacylation reaction was observed for a 21-residues deletion mutant of the C-terminal end. These results show that the genetic approach of the three hybrid system is widely applicable and is a powerful tool for the investigation of tRNA:synthetase interactions.
Théobald-Dietrich A, Frugier M, Giege R, Rudinger-Thirion J
Atypical archaeal tRNA pyrrolysine transcript behaves towards EF-Tu as a typical elongator tRNA Journal Article
In: Nucleic Acids Res, vol. 32, no. 3, pp. 1091-1096, 2004, ISBN: 14872064, (1362-4962 Journal Article).
Abstract | Links | BibTeX | Tags: Anticodon/metabolism Base Sequence Lysine/*analogs & derivatives/*metabolism Lysine-tRNA Ligase/metabolism Methanosarcina barkeri/genetics Mitochondria/genetics Molecular Sequence Data Nucleic Acid Conformation Peptide Elongation Factor Tu/*metabolism RNA, Archaeal/chemistry/*metabolism RNA, FRUGIER, Non-U.S. Gov't Yeasts/enzymology, Ser/chemistry Selenocysteine/metabolism Support, Transfer, Transfer/chemistry/*metabolism RNA, Unité ARN
@article{,
title = {Atypical archaeal tRNA pyrrolysine transcript behaves towards EF-Tu as a typical elongator tRNA},
author = {A Théobald-Dietrich and M Frugier and R Giege and J Rudinger-Thirion},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14872064},
isbn = {14872064},
year = {2004},
date = {2004-01-01},
journal = {Nucleic Acids Res},
volume = {32},
number = {3},
pages = {1091-1096},
abstract = {The newly discovered tRNA(Pyl) is involved in specific incorporation of pyrrolysine in the active site of methylamine methyltransferases in the archaeon Methanosarcina barkeri. In solution probing experiments, a transcript derived from tRNA(Pyl) displays a secondary fold slightly different from the canonical cloverleaf and interestingly similar to that of bovine mitochondrial tRNA(Ser)(uga). Aminoacylation of tRNA(Pyl) transcript by a typical class II synthetase, LysRS from yeast, was possible when its amber anticodon CUA was mutated into a lysine UUU anticodon. Hydrolysis protection assays show that lysylated tRNA(Pyl) can be recognized by bacterial elongation factor. This indicates that no antideterminant sequence is present in the body of the tRNA(Pyl) transcript to prevent it from interacting with EF-Tu, in contrast with the otherwise functionally similar tRNA(Sec) that mediates selenocysteine incorporation.},
note = {1362-4962
Journal Article},
keywords = {Anticodon/metabolism Base Sequence Lysine/*analogs & derivatives/*metabolism Lysine-tRNA Ligase/metabolism Methanosarcina barkeri/genetics Mitochondria/genetics Molecular Sequence Data Nucleic Acid Conformation Peptide Elongation Factor Tu/*metabolism RNA, Archaeal/chemistry/*metabolism RNA, FRUGIER, Non-U.S. Gov't Yeasts/enzymology, Ser/chemistry Selenocysteine/metabolism Support, Transfer, Transfer/chemistry/*metabolism RNA, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The newly discovered tRNA(Pyl) is involved in specific incorporation of pyrrolysine in the active site of methylamine methyltransferases in the archaeon Methanosarcina barkeri. In solution probing experiments, a transcript derived from tRNA(Pyl) displays a secondary fold slightly different from the canonical cloverleaf and interestingly similar to that of bovine mitochondrial tRNA(Ser)(uga). Aminoacylation of tRNA(Pyl) transcript by a typical class II synthetase, LysRS from yeast, was possible when its amber anticodon CUA was mutated into a lysine UUU anticodon. Hydrolysis protection assays show that lysylated tRNA(Pyl) can be recognized by bacterial elongation factor. This indicates that no antideterminant sequence is present in the body of the tRNA(Pyl) transcript to prevent it from interacting with EF-Tu, in contrast with the otherwise functionally similar tRNA(Sec) that mediates selenocysteine incorporation.
Sohm B, Sissler M, Park H, King M P, Florentz C
Recognition of human mitochondrial tRNALeu(UUR) by its cognate leucyl-tRNA synthetase Journal Article
In: J Mol Biol, vol. 339, no. 1, pp. 17-29, 2004, ISBN: 15123417, (0022-2836 Journal Article).
Abstract | Links | BibTeX | Tags: Cultured, FLORENTZ, FLORENTZ *Acylation Base Sequence Comparative Study Human Kinetics Leucine/metabolism Leucine-tRNA Ligase/genetics/*metabolism Mitochondria/*metabolism Molecular Sequence Data Mutation Nucleic Acid Conformation Osteosarcoma/metabolism RNA/*genetics/metabolism RNA, Genetic/*genetics Tumor Cells, Leu/genetics/*metabolism Solutions Substrate Specificity Support, Non-U.S. Gov't Support, P.H.S. Transcription, SISSLER, Transfer, U.S. Gov't, Unité ARN
@article{,
title = {Recognition of human mitochondrial tRNALeu(UUR) by its cognate leucyl-tRNA synthetase},
author = {B Sohm and M Sissler and H Park and M P King and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15123417},
isbn = {15123417},
year = {2004},
date = {2004-01-01},
journal = {J Mol Biol},
volume = {339},
number = {1},
pages = {17-29},
abstract = {Accuracy of protein synthesis depends on specific recognition and aminoacylation of tRNAs by their cognate aminoacyl-tRNA synthetases. Rules governing these processes have been established for numerous prokaryotic and eukaryotic cytoplasmic systems, but only limited information is available for human mitochondrial systems. It has been shown that the in vitro transcribed human mitochondrial tRNA(Leu(UUR)) does not fold into the expected cloverleaf, but is however aminoacylated by the human mitochondrial leucyl-tRNA synthetase. Here, the role of the structure of the amino acid acceptor branch and the anticodon branch of tRNA(Leu(UUR)) in recognition by leucyl-tRNA synthetase was investigated. The kinetic parameters for aminoacylation of wild-type and mutant tRNA(Leu(UUR)) transcripts and of native tRNA(Leu(UUR)) were determined. Solution structure probing was performed in the presence or in the absence of leucyl-tRNA synthetase and correlated with the aminoacylation kinetics for each tRNA. Replacement of mismatches in either the anticodon-stem or D-stem that are present in the wild-type tRNA(Leu(UUR)) by G-C base-pairs is sufficient to induce (i) cloverleaf folding, (ii) improved aminoacylation efficiency, and (iii) interactions with the synthetase that are similar to those with the native tRNA(Leu(UUR)). Leucyl-tRNA synthetase contacts tRNA(Leu(UUR)) in the amino acid acceptor stem, the anticodon stem, and the D-loop, which is unprecedented for a leucine aminoacylation system.},
note = {0022-2836
Journal Article},
keywords = {Cultured, FLORENTZ, FLORENTZ *Acylation Base Sequence Comparative Study Human Kinetics Leucine/metabolism Leucine-tRNA Ligase/genetics/*metabolism Mitochondria/*metabolism Molecular Sequence Data Mutation Nucleic Acid Conformation Osteosarcoma/metabolism RNA/*genetics/metabolism RNA, Genetic/*genetics Tumor Cells, Leu/genetics/*metabolism Solutions Substrate Specificity Support, Non-U.S. Gov't Support, P.H.S. Transcription, SISSLER, Transfer, U.S. Gov't, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Accuracy of protein synthesis depends on specific recognition and aminoacylation of tRNAs by their cognate aminoacyl-tRNA synthetases. Rules governing these processes have been established for numerous prokaryotic and eukaryotic cytoplasmic systems, but only limited information is available for human mitochondrial systems. It has been shown that the in vitro transcribed human mitochondrial tRNA(Leu(UUR)) does not fold into the expected cloverleaf, but is however aminoacylated by the human mitochondrial leucyl-tRNA synthetase. Here, the role of the structure of the amino acid acceptor branch and the anticodon branch of tRNA(Leu(UUR)) in recognition by leucyl-tRNA synthetase was investigated. The kinetic parameters for aminoacylation of wild-type and mutant tRNA(Leu(UUR)) transcripts and of native tRNA(Leu(UUR)) were determined. Solution structure probing was performed in the presence or in the absence of leucyl-tRNA synthetase and correlated with the aminoacylation kinetics for each tRNA. Replacement of mismatches in either the anticodon-stem or D-stem that are present in the wild-type tRNA(Leu(UUR)) by G-C base-pairs is sufficient to induce (i) cloverleaf folding, (ii) improved aminoacylation efficiency, and (iii) interactions with the synthetase that are similar to those with the native tRNA(Leu(UUR)). Leucyl-tRNA synthetase contacts tRNA(Leu(UUR)) in the amino acid acceptor stem, the anticodon stem, and the D-loop, which is unprecedented for a leucine aminoacylation system.
Sissler M, Helm M, Frugier M, Giege R, Florentz C
Aminoacylation properties of pathology-related human mitochondrial tRNA(Lys) variants Journal Article
In: RNA, vol. 10, no. 5, pp. 841-853, 2004, ISBN: 15100439, (1355-8382 Journal Article).
Abstract | Links | BibTeX | Tags: ERIANI, FLORENTZ, FLORENTZ GIEGE Acylation Aminoacyltransferases/*genetics Human MERRF Syndrome/genetics Mitochondria/*genetics Mitochondrial Diseases/*genetics Mutation Nucleic Acid Conformation RNA, FRUGIER, Lys/*genetics Sequence Analysis, Non-U.S. Gov't Variation (Genetics), RNA Support, SISSLER, Transfer, Unité ARN
@article{,
title = {Aminoacylation properties of pathology-related human mitochondrial tRNA(Lys) variants},
author = {M Sissler and M Helm and M Frugier and R Giege and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15100439},
isbn = {15100439},
year = {2004},
date = {2004-01-01},
journal = {RNA},
volume = {10},
number = {5},
pages = {841-853},
abstract = {In vitro transcription has proven to be a successful tool for preparation of functional RNAs, especially in the tRNA field, in which, despite the absence of post-transcriptional modifications, transcripts are correctly folded and functionally active. Human mitochondrial (mt) tRNA(Lys) deviates from this principle and folds into various inactive conformations, due to the absence of the post-transcriptional modification m(1)A9 which hinders base-pairing with U64 in the native tRNA. Unavailability of a functional transcript is a serious drawback for structure/function investigations as well as in deciphering the molecular mechanisms by which point mutations in the mt tRNA(Lys) gene cause severe human disorders. Here, we show that an engineered in vitro transcribed "pseudo-WT" tRNA(Lys) variant is efficiently recognized by lysyl-tRNA synthetase and can substitute for the WT tRNA as a valuable reference molecule. This has been exploited in a systematic analysis of the effects on aminoacylation of nine pathology-related mutations described so far. The sole mutation located in a loop of the tRNA secondary structure, A8344G, does not affect aminoacylation efficiency. Out of eight mutations located in helical domains converting canonical Watson-Crick pairs into G-U pairs or C.A mismatches, six have no effect on aminoacylation (A8296G, U8316C, G8342A, U8356C, U8362G, G8363A), and two lead to drastic decreases (5000- to 7000-fold) in lysylation efficiencies (G8313A and G8328A). This screening, allowing for analysis of the primary impact level of all mutations affecting one tRNA under comparable conditions, indicates distinct molecular origins for different disorders.},
note = {1355-8382
Journal Article},
keywords = {ERIANI, FLORENTZ, FLORENTZ GIEGE Acylation Aminoacyltransferases/*genetics Human MERRF Syndrome/genetics Mitochondria/*genetics Mitochondrial Diseases/*genetics Mutation Nucleic Acid Conformation RNA, FRUGIER, Lys/*genetics Sequence Analysis, Non-U.S. Gov't Variation (Genetics), RNA Support, SISSLER, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In vitro transcription has proven to be a successful tool for preparation of functional RNAs, especially in the tRNA field, in which, despite the absence of post-transcriptional modifications, transcripts are correctly folded and functionally active. Human mitochondrial (mt) tRNA(Lys) deviates from this principle and folds into various inactive conformations, due to the absence of the post-transcriptional modification m(1)A9 which hinders base-pairing with U64 in the native tRNA. Unavailability of a functional transcript is a serious drawback for structure/function investigations as well as in deciphering the molecular mechanisms by which point mutations in the mt tRNA(Lys) gene cause severe human disorders. Here, we show that an engineered in vitro transcribed "pseudo-WT" tRNA(Lys) variant is efficiently recognized by lysyl-tRNA synthetase and can substitute for the WT tRNA as a valuable reference molecule. This has been exploited in a systematic analysis of the effects on aminoacylation of nine pathology-related mutations described so far. The sole mutation located in a loop of the tRNA secondary structure, A8344G, does not affect aminoacylation efficiency. Out of eight mutations located in helical domains converting canonical Watson-Crick pairs into G-U pairs or C.A mismatches, six have no effect on aminoacylation (A8296G, U8316C, G8342A, U8356C, U8362G, G8363A), and two lead to drastic decreases (5000- to 7000-fold) in lysylation efficiencies (G8313A and G8328A). This screening, allowing for analysis of the primary impact level of all mutations affecting one tRNA under comparable conditions, indicates distinct molecular origins for different disorders.
Rangan P, Masquida B, Westhof E, Woodson S A
Architecture and folding mechanism of the Azoarcus Group I Pre-tRNA Journal Article
In: J Mol Biol, vol. 339, no. 1, pp. 41-51, 2004, ISBN: 15123419, (0022-2836 Journal Article).
Abstract | Links | BibTeX | Tags: Azoarcus/enzymology/*genetics Base Sequence Binding Sites Exoribonucleases/metabolism Hydroxyl Radical/metabolism Introns/*genetics Magnesium/chemistry Models, Bacterial/*chemistry/genetics/*metabolism RNA, Ile/chemistry/*genetics Substrate Specificity Support, Molecular Molecular Sequence Data *Nucleic Acid Conformation RNA Precursors/*genetics RNA Splice Sites/genetics RNA Splicing RNA, Non-U.S. Gov't Support, P.H.S., Transfer, U.S. Gov't, Unité ARN, WESTHOF
@article{,
title = {Architecture and folding mechanism of the Azoarcus Group I Pre-tRNA},
author = {P Rangan and B Masquida and E Westhof and S A Woodson},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15123419},
isbn = {15123419},
year = {2004},
date = {2004-01-01},
journal = {J Mol Biol},
volume = {339},
number = {1},
pages = {41-51},
abstract = {Self-splicing RNAs must evolve to function in their specific exon context. The conformation of a group I pre-tRNA(ile) from the bacterium Azoarcus was probed by ribonuclease T(1) and hydroxyl radical cleavage, and by native gel electrophoresis. Biochemical data and three-dimensional models of the pre-tRNA showed that the tRNA is folded, and that the tRNA and intron sequences form separate tertiary domains. Models of the active site before steps 1 and 2 of the splicing reaction predict that exchange of the external G-cofactor and the 3'-terminal G is accomplished by a slight conformational change in P9.0 of the Azoarcus group I intron. Kinetic assays showed that the pre-tRNA folds in minutes, much more slowly than the intron alone. The dependence of the folding kinetics on Mg(2+) and the concentration of urea, and RNase T(1) experiments showed that formation of native pre-tRNA is delayed by misfolding of P3-P9, including mispairing between residues in P9 and the tRNA. Thus, although the intron and tRNA sequences form separate domains in the native pre-tRNA, their folding is coupled via metastable non-native base-pairs. This could help prevent premature processing of the 5' and 3' ends of unspliced pre-tRNA.},
note = {0022-2836
Journal Article},
keywords = {Azoarcus/enzymology/*genetics Base Sequence Binding Sites Exoribonucleases/metabolism Hydroxyl Radical/metabolism Introns/*genetics Magnesium/chemistry Models, Bacterial/*chemistry/genetics/*metabolism RNA, Ile/chemistry/*genetics Substrate Specificity Support, Molecular Molecular Sequence Data *Nucleic Acid Conformation RNA Precursors/*genetics RNA Splice Sites/genetics RNA Splicing RNA, Non-U.S. Gov't Support, P.H.S., Transfer, U.S. Gov't, Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
Self-splicing RNAs must evolve to function in their specific exon context. The conformation of a group I pre-tRNA(ile) from the bacterium Azoarcus was probed by ribonuclease T(1) and hydroxyl radical cleavage, and by native gel electrophoresis. Biochemical data and three-dimensional models of the pre-tRNA showed that the tRNA is folded, and that the tRNA and intron sequences form separate tertiary domains. Models of the active site before steps 1 and 2 of the splicing reaction predict that exchange of the external G-cofactor and the 3'-terminal G is accomplished by a slight conformational change in P9.0 of the Azoarcus group I intron. Kinetic assays showed that the pre-tRNA folds in minutes, much more slowly than the intron alone. The dependence of the folding kinetics on Mg(2+) and the concentration of urea, and RNase T(1) experiments showed that formation of native pre-tRNA is delayed by misfolding of P3-P9, including mispairing between residues in P9 and the tRNA. Thus, although the intron and tRNA sequences form separate domains in the native pre-tRNA, their folding is coupled via metastable non-native base-pairs. This could help prevent premature processing of the 5' and 3' ends of unspliced pre-tRNA.
Martin F, Barends S, Eriani G
Single amino acid changes in AspRS reveal alternative routes for expanding its tRNA repertoire in vivo Journal Article
In: Nucleic Acids Res, vol. 32, no. 13, pp. 4081-4089, 2004, ISBN: 15289581, (1362-4962 Journal Article).
Abstract | Links | BibTeX | Tags: Amino Acid Substitution Anticodon/metabolism Aspartate-tRNA Ligase/*chemistry/genetics/*metabolism Aspartic Acid/metabolism Binding Sites Models, Amino Acyl/chemistry/*metabolism Support, ERIANI, Molecular Mutation Phenotype Protein Engineering Protein Structure, Non-U.S. Gov't, Tertiary RNA, Transfer, Unité ARN
@article{,
title = {Single amino acid changes in AspRS reveal alternative routes for expanding its tRNA repertoire in vivo},
author = {F Martin and S Barends and G Eriani},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15289581},
isbn = {15289581},
year = {2004},
date = {2004-01-01},
journal = {Nucleic Acids Res},
volume = {32},
number = {13},
pages = {4081-4089},
abstract = {Aminoacyl-tRNA synthetases (aaRSs) are enzymes that are highly specific for their tRNA substrates. Here, we describe the expansion of a class IIb aaRS-tRNA specificity by a genetic selection that involves the use of a modified tRNA displaying an amber anticodon and the argE(amber) and lacZ(amber) reporters. The study was performed on Escherichia coli aspartyl-tRNA synthetase (AspRS) and amber tRNA(Asp). Nine AspRS mutants able to charge the amber tRNA(Asp) and to suppress the reporter genes were selected from a randomly mutated library. All the mutants exhibited a new amber tRNA(Asp) specificity in addition to the initial native tRNA(Asp). Six mutations were found in the anticodon-binding site located in the N-terminal OB-fold. The strongest suppressor was a mutation of residue Glu-93 that contacts specifically the anticodon nucleotide 34 in the crystal structure. The other mutations in the OB-fold were found at close distance from the anticodon in the so-called loop L45 and strand S1. They concern residues that do not contact tRNA(Asp) in the native complex. In addition, this study shows that suppressors can carry mutations located far from the anticodon-binding site. One such mutation was found in the synthetase hinge-module where it increases the tRNA(Asp)-charging rate, and two other mutations were found in the prokaryotic-specific insertion domain and the catalytic core. These mutants seem to act by indirect effects on the tRNA acceptor stem binding and on the conformation of the active site of the enzyme. Altogether, these data suggest the existence of various ways for modifying the mechanism of tRNA discrimination.},
note = {1362-4962
Journal Article},
keywords = {Amino Acid Substitution Anticodon/metabolism Aspartate-tRNA Ligase/*chemistry/genetics/*metabolism Aspartic Acid/metabolism Binding Sites Models, Amino Acyl/chemistry/*metabolism Support, ERIANI, Molecular Mutation Phenotype Protein Engineering Protein Structure, Non-U.S. Gov't, Tertiary RNA, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Aminoacyl-tRNA synthetases (aaRSs) are enzymes that are highly specific for their tRNA substrates. Here, we describe the expansion of a class IIb aaRS-tRNA specificity by a genetic selection that involves the use of a modified tRNA displaying an amber anticodon and the argE(amber) and lacZ(amber) reporters. The study was performed on Escherichia coli aspartyl-tRNA synthetase (AspRS) and amber tRNA(Asp). Nine AspRS mutants able to charge the amber tRNA(Asp) and to suppress the reporter genes were selected from a randomly mutated library. All the mutants exhibited a new amber tRNA(Asp) specificity in addition to the initial native tRNA(Asp). Six mutations were found in the anticodon-binding site located in the N-terminal OB-fold. The strongest suppressor was a mutation of residue Glu-93 that contacts specifically the anticodon nucleotide 34 in the crystal structure. The other mutations in the OB-fold were found at close distance from the anticodon in the so-called loop L45 and strand S1. They concern residues that do not contact tRNA(Asp) in the native complex. In addition, this study shows that suppressors can carry mutations located far from the anticodon-binding site. One such mutation was found in the synthetase hinge-module where it increases the tRNA(Asp)-charging rate, and two other mutations were found in the prokaryotic-specific insertion domain and the catalytic core. These mutants seem to act by indirect effects on the tRNA acceptor stem binding and on the conformation of the active site of the enzyme. Altogether, these data suggest the existence of various ways for modifying the mechanism of tRNA discrimination.
Levinger L, Oestreich I, Florentz C, Morl M
A pathogenesis-associated mutation in human mitochondrial tRNALeu(UUR) leads to reduced 3'-end processing and CCA addition Journal Article
In: J Mol Biol, vol. 337, no. 3, pp. 535-544, 2004, ISBN: 15019775, (0022-2836 Journal Article).
Abstract | Links | BibTeX | Tags: FLORENTZ, FLORENTZ Human Kinetics Mitochondrial Diseases/*genetics Nucleic Acid Conformation *Point Mutation RNA/*genetics/physiology *RNA 3' End Processing RNA Nucleotidyltransferases/metabolism RNA, Leu/*genetics/physiology Support, Non-U.S. Gov't Support, P.H.S., Transfer, U.S. Gov't, Unité ARN
@article{,
title = {A pathogenesis-associated mutation in human mitochondrial tRNALeu(UUR) leads to reduced 3'-end processing and CCA addition},
author = {L Levinger and I Oestreich and C Florentz and M Morl},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15019775},
isbn = {15019775},
year = {2004},
date = {2004-01-01},
journal = {J Mol Biol},
volume = {337},
number = {3},
pages = {535-544},
abstract = {Point mutations in mitochondrial tRNAs can cause severe multisystemic disorders such as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and myoclonus epilepsy with ragged-red fibers (MERRF). Some of these mutations impair one or more steps of tRNA maturation and protein biosynthesis including 5'-end-processing, post-transcriptional base modification, structural stability, aminoacylation, and formation of tRNA-ribosomal complexes. tRNALeu(UUR), an etiologic hot spot for such diseases, harbors 20 of more than 90 disease-associated mutations described to date. Here, the pathogenesis-associated base substitutions A3243G, T3250C, T3271C, A3302G and C3303T within this tRNA were tested for their effects on endonucleolytic 3'-end processing and CCA addition at the tRNA 3'-terminus. Whereas mutations A3243G, A3302G and C3303T reduced the efficiency of 3'-end cleavage, only the C3303T substitution was a less efficient substrate for CCA addition. These results support the view that pathogenesis may be elicited through cumulative effects of tRNA mutations: a mutation can impede several pre-tRNA processing steps, with each such reduction contributing to the overall impairment of tRNA function.},
note = {0022-2836
Journal Article},
keywords = {FLORENTZ, FLORENTZ Human Kinetics Mitochondrial Diseases/*genetics Nucleic Acid Conformation *Point Mutation RNA/*genetics/physiology *RNA 3' End Processing RNA Nucleotidyltransferases/metabolism RNA, Leu/*genetics/physiology Support, Non-U.S. Gov't Support, P.H.S., Transfer, U.S. Gov't, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Point mutations in mitochondrial tRNAs can cause severe multisystemic disorders such as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and myoclonus epilepsy with ragged-red fibers (MERRF). Some of these mutations impair one or more steps of tRNA maturation and protein biosynthesis including 5'-end-processing, post-transcriptional base modification, structural stability, aminoacylation, and formation of tRNA-ribosomal complexes. tRNALeu(UUR), an etiologic hot spot for such diseases, harbors 20 of more than 90 disease-associated mutations described to date. Here, the pathogenesis-associated base substitutions A3243G, T3250C, T3271C, A3302G and C3303T within this tRNA were tested for their effects on endonucleolytic 3'-end processing and CCA addition at the tRNA 3'-terminus. Whereas mutations A3243G, A3302G and C3303T reduced the efficiency of 3'-end cleavage, only the C3303T substitution was a less efficient substrate for CCA addition. These results support the view that pathogenesis may be elicited through cumulative effects of tRNA mutations: a mutation can impede several pre-tRNA processing steps, with each such reduction contributing to the overall impairment of tRNA function.
Fender A, Sissler M, Florentz C, Giege R
Functional idiosyncrasies of tRNA isoacceptors in cognate and noncognate aminoacylation systems Journal Article
In: Biochimie, vol. 86, no. 1, pp. 21-29, 2004, ISBN: 14987797, (0300-9084 Journal Article).
Abstract | Links | BibTeX | Tags: Amino Acyl/genetics/*metabolism Saccharomyces cerevisiae Substrate Specificity/genetics/physiology Support, Chemical Molecular Sequence Data Mutation Nucleic Acid Conformation Protein Binding/physiology RNA, FLORENTZ, GIEGE FLORENTZ Amino Acid Activation/physiology Amino Acyl-tRNA Ligases/*metabolism Anticodon Bacterial Proteins/metabolism Base Sequence Cloning, Molecular Computer Simulation Escherichia coli Models, Non-U.S. Gov't Thermus thermophilus, SISSLER, Transfer, Transfer/genetics/*metabolism RNA, Unité ARN
@article{,
title = {Functional idiosyncrasies of tRNA isoacceptors in cognate and noncognate aminoacylation systems},
author = {A Fender and M Sissler and C Florentz and R Giege},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14987797},
isbn = {14987797},
year = {2004},
date = {2004-01-01},
journal = {Biochimie},
volume = {86},
number = {1},
pages = {21-29},
abstract = {The specificity of transfer RNA aminoacylation by cognate aminoacyl-tRNA synthetase is a crucial step for synthesis of functional proteins. It is established that the aminoacylation identity of a single tRNA or of a family of tRNA isoacceptors is linked to the presence of positive signals (determinants) allowing recognition by cognate synthetases and negative signals (antideterminants) leading to rejection by the noncognate ones. The completion of identity sets was generally tested by transplantation of the corresponding nucleotides into one or several host tRNAs which acquire as a consequence the new aminoacylation specificities. Such transplantation experiments were also useful to detect peculiar structural refinements required for optimal expression of a given aminoacylation identity set within a host tRNA. This study explores expression of the defined yeast aspartate identity set into different tRNA scaffolds of a same specificity, namely the four yeast tRNA(Arg) isoacceptors. The goal was to investigate whether expression of the new identity is similar due to the unique specificity of the host tRNAs or whether it is differently expressed due to their peculiar sequences and structural features. In vitro transcribed native tRNA(Arg) isoacceptors and variants bearing the aspartate identity elements were prepared and their aminoacylation properties established. The four wild-type isoacceptors are active in arginylation with catalytic efficiencies in a 20-fold range and are inactive in aspartylation. While transplanted tRNA(1)(Arg) and tRNA(4)(Arg) are converted into highly efficient substrates for yeast aspartyl-tRNA synthetase, transplanted tRNA(2)(Arg) and tRNA(3)(Arg) remain poorly aspartylated. Search for antideterminants in these two tRNAs reveals idiosyncratic features. Conversion of the single base-pair C6-G67 into G6-C67, the pair present in tRNA(Asp), allows full expression of the aspartate identity in the transplanted tRNA(2)(Arg), but not in tRNA(3)(Arg). It is concluded that the different isoacceptor tRNAs protect themselves from misaminoacylation by idiosyncratic pathways of antidetermination.},
note = {0300-9084
Journal Article},
keywords = {Amino Acyl/genetics/*metabolism Saccharomyces cerevisiae Substrate Specificity/genetics/physiology Support, Chemical Molecular Sequence Data Mutation Nucleic Acid Conformation Protein Binding/physiology RNA, FLORENTZ, GIEGE FLORENTZ Amino Acid Activation/physiology Amino Acyl-tRNA Ligases/*metabolism Anticodon Bacterial Proteins/metabolism Base Sequence Cloning, Molecular Computer Simulation Escherichia coli Models, Non-U.S. Gov't Thermus thermophilus, SISSLER, Transfer, Transfer/genetics/*metabolism RNA, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The specificity of transfer RNA aminoacylation by cognate aminoacyl-tRNA synthetase is a crucial step for synthesis of functional proteins. It is established that the aminoacylation identity of a single tRNA or of a family of tRNA isoacceptors is linked to the presence of positive signals (determinants) allowing recognition by cognate synthetases and negative signals (antideterminants) leading to rejection by the noncognate ones. The completion of identity sets was generally tested by transplantation of the corresponding nucleotides into one or several host tRNAs which acquire as a consequence the new aminoacylation specificities. Such transplantation experiments were also useful to detect peculiar structural refinements required for optimal expression of a given aminoacylation identity set within a host tRNA. This study explores expression of the defined yeast aspartate identity set into different tRNA scaffolds of a same specificity, namely the four yeast tRNA(Arg) isoacceptors. The goal was to investigate whether expression of the new identity is similar due to the unique specificity of the host tRNAs or whether it is differently expressed due to their peculiar sequences and structural features. In vitro transcribed native tRNA(Arg) isoacceptors and variants bearing the aspartate identity elements were prepared and their aminoacylation properties established. The four wild-type isoacceptors are active in arginylation with catalytic efficiencies in a 20-fold range and are inactive in aspartylation. While transplanted tRNA(1)(Arg) and tRNA(4)(Arg) are converted into highly efficient substrates for yeast aspartyl-tRNA synthetase, transplanted tRNA(2)(Arg) and tRNA(3)(Arg) remain poorly aspartylated. Search for antideterminants in these two tRNAs reveals idiosyncratic features. Conversion of the single base-pair C6-G67 into G6-C67, the pair present in tRNA(Asp), allows full expression of the aspartate identity in the transplanted tRNA(2)(Arg), but not in tRNA(3)(Arg). It is concluded that the different isoacceptor tRNAs protect themselves from misaminoacylation by idiosyncratic pathways of antidetermination.
Fender A, Geslain R, Eriani G, Giege R, Sissler M, Florentz C
A yeast arginine specific tRNA is a remnant aspartate acceptor Journal Article
In: Nucleic Acids Res, vol. 32, no. 17, pp. 5076-5086, 2004, ISBN: 15452274, (1362-4962 Journal Article).
Abstract | Links | BibTeX | Tags: Arg/*chemistry/genetics/metabolism RNA, Asp/*chemistry/genetics/metabolism Saccharomyces cerevisiae/*genetics Sequence Alignment Support, ERIANI, ERIANI FLORENTZ GIEGE Aspartic Acid/metabolism Base Sequence *Evolution, FLORENTZ, Fungal/*chemistry/genetics/metabolism RNA, Molecular Molecular Sequence Data Point Mutation RNA, Non-U.S. Gov't, SISSLER, Transfer, Unité ARN
@article{,
title = {A yeast arginine specific tRNA is a remnant aspartate acceptor},
author = {A Fender and R Geslain and G Eriani and R Giege and M Sissler and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15452274},
isbn = {15452274},
year = {2004},
date = {2004-01-01},
journal = {Nucleic Acids Res},
volume = {32},
number = {17},
pages = {5076-5086},
abstract = {High specificity in aminoacylation of transfer RNAs (tRNAs) with the help of their cognate aminoacyl-tRNA synthetases (aaRSs) is a guarantee for accurate genetic translation. Structural and mechanistic peculiarities between the different tRNA/aaRS couples, suggest that aminoacylation systems are unrelated. However, occurrence of tRNA mischarging by non-cognate aaRSs reflects the relationship between such systems. In Saccharomyces cerevisiae, functional links between arginylation and aspartylation systems have been reported. In particular, it was found that an in vitro transcribed tRNAAsp is a very efficient substrate for ArgRS. In this study, the relationship of arginine and aspartate systems is further explored, based on the discovery of a fourth isoacceptor in the yeast genome, tRNA4Arg. This tRNA has a sequence strikingly similar to that of tRNAAsp but distinct from those of the other three arginine isoacceptors. After transplantation of the full set of aspartate identity elements into the four arginine isoacceptors, tRNA4Arg gains the highest aspartylation efficiency. Moreover, it is possible to convert tRNA4Arg into an aspartate acceptor, as efficient as tRNAAsp, by only two point mutations, C38 and G73, despite the absence of the major anticodon aspartate identity elements. Thus, cryptic aspartate identity elements are embedded within tRNA4Arg. The latent aspartate acceptor capacity in a contemporary tRNAArg leads to the proposal of an evolutionary link between tRNA4Arg and tRNAAsp genes.},
note = {1362-4962
Journal Article},
keywords = {Arg/*chemistry/genetics/metabolism RNA, Asp/*chemistry/genetics/metabolism Saccharomyces cerevisiae/*genetics Sequence Alignment Support, ERIANI, ERIANI FLORENTZ GIEGE Aspartic Acid/metabolism Base Sequence *Evolution, FLORENTZ, Fungal/*chemistry/genetics/metabolism RNA, Molecular Molecular Sequence Data Point Mutation RNA, Non-U.S. Gov't, SISSLER, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
High specificity in aminoacylation of transfer RNAs (tRNAs) with the help of their cognate aminoacyl-tRNA synthetases (aaRSs) is a guarantee for accurate genetic translation. Structural and mechanistic peculiarities between the different tRNA/aaRS couples, suggest that aminoacylation systems are unrelated. However, occurrence of tRNA mischarging by non-cognate aaRSs reflects the relationship between such systems. In Saccharomyces cerevisiae, functional links between arginylation and aspartylation systems have been reported. In particular, it was found that an in vitro transcribed tRNAAsp is a very efficient substrate for ArgRS. In this study, the relationship of arginine and aspartate systems is further explored, based on the discovery of a fourth isoacceptor in the yeast genome, tRNA4Arg. This tRNA has a sequence strikingly similar to that of tRNAAsp but distinct from those of the other three arginine isoacceptors. After transplantation of the full set of aspartate identity elements into the four arginine isoacceptors, tRNA4Arg gains the highest aspartylation efficiency. Moreover, it is possible to convert tRNA4Arg into an aspartate acceptor, as efficient as tRNAAsp, by only two point mutations, C38 and G73, despite the absence of the major anticodon aspartate identity elements. Thus, cryptic aspartate identity elements are embedded within tRNA4Arg. The latent aspartate acceptor capacity in a contemporary tRNAArg leads to the proposal of an evolutionary link between tRNA4Arg and tRNAAsp genes.
Dubois D Y, Blaise M, Becker H D, Campanacci V, Keith G, Giege R, Cambillau C, Lapointe J, Kern D
An aminoacyl-tRNA synthetase-like protein encoded by the Escherichia coli yadB gene glutamylates specifically tRNAAsp Journal Article
In: Proc Natl Acad Sci U S A, vol. 101, no. 20, pp. 7530-7535, 2004, ISBN: 15096594, (0027-8424 Journal Article).
Abstract | Links | BibTeX | Tags: Asp/*metabolism Support, KERN GIEGE Amino Acyl-tRNA Ligases/chemistry/genetics/*metabolism Crystallography, Messenger/metabolism RNA, Non-U.S. Gov't, Tertiary RNA, Transfer, Unité ARN, X-Ray Escherichia coli/enzymology/genetics/*metabolism Escherichia coli Proteins/chemistry/genetics/*metabolism Glutamic Acid/*metabolism Peptide Elongation Factor 2/metabolism Phylogeny Protein Structure
@article{,
title = {An aminoacyl-tRNA synthetase-like protein encoded by the Escherichia coli yadB gene glutamylates specifically tRNAAsp},
author = {D Y Dubois and M Blaise and H D Becker and V Campanacci and G Keith and R Giege and C Cambillau and J Lapointe and D Kern},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15096594},
isbn = {15096594},
year = {2004},
date = {2004-01-01},
journal = {Proc Natl Acad Sci U S A},
volume = {101},
number = {20},
pages = {7530-7535},
abstract = {The product of the Escherichia coli yadB gene is homologous to the N-terminal part of bacterial glutamyl-tRNA synthetases (GluRSs), including the Rossmann fold with the acceptor-binding domain and the stem-contact fold. This GluRS-like protein, which lacks the anticodon-binding domain, does not use tRNA(Glu) as substrate in vitro nor in vivo, but aminoacylates tRNA(Asp) with glutamate. The yadB gene is expressed in wild-type E. coli as an operon with the dksA gene, which encodes a protein involved in the general stress response by means of its action at the translational level. The fate of the glutamylated tRNA(Asp) is not known, but its incapacity to bind elongation factor Tu suggests that it is not involved in ribosomal protein synthesis. Genes homologous to yadB are present only in bacteria, mostly in Proteobacteria. Sequence alignments and phylogenetic analyses show that the YadB proteins form a distinct monophyletic group related to the bacterial and organellar GluRSs (alpha-type GlxRSs superfamily) with ubiquitous function as suggested by the similar functional properties of the YadB homologue from Neisseria meningitidis.},
note = {0027-8424
Journal Article},
keywords = {Asp/*metabolism Support, KERN GIEGE Amino Acyl-tRNA Ligases/chemistry/genetics/*metabolism Crystallography, Messenger/metabolism RNA, Non-U.S. Gov't, Tertiary RNA, Transfer, Unité ARN, X-Ray Escherichia coli/enzymology/genetics/*metabolism Escherichia coli Proteins/chemistry/genetics/*metabolism Glutamic Acid/*metabolism Peptide Elongation Factor 2/metabolism Phylogeny Protein Structure},
pubstate = {published},
tppubtype = {article}
}
The product of the Escherichia coli yadB gene is homologous to the N-terminal part of bacterial glutamyl-tRNA synthetases (GluRSs), including the Rossmann fold with the acceptor-binding domain and the stem-contact fold. This GluRS-like protein, which lacks the anticodon-binding domain, does not use tRNA(Glu) as substrate in vitro nor in vivo, but aminoacylates tRNA(Asp) with glutamate. The yadB gene is expressed in wild-type E. coli as an operon with the dksA gene, which encodes a protein involved in the general stress response by means of its action at the translational level. The fate of the glutamylated tRNA(Asp) is not known, but its incapacity to bind elongation factor Tu suggests that it is not involved in ribosomal protein synthesis. Genes homologous to yadB are present only in bacteria, mostly in Proteobacteria. Sequence alignments and phylogenetic analyses show that the YadB proteins form a distinct monophyletic group related to the bacterial and organellar GluRSs (alpha-type GlxRSs superfamily) with ubiquitous function as suggested by the similar functional properties of the YadB homologue from Neisseria meningitidis.
Costa A, de Barros J P Pais, Keith G, Baranowski W, Desgres J
In: J Chromatogr B Analyt Technol Biomed Life Sci, vol. 801, no. 2, pp. 237-247, 2004, ISBN: 14751792, (1570-0232 Journal Article).
Abstract | Links | BibTeX | Tags: Amino Acyl/chemistry RNA, Asn/chemistry Rats Support, Cultured, Cultured Chickens *Chromatography, Experimental Nucleoside Q/*analogs & derivatives/*analysis RNA, High Pressure Liquid Hepatocytes/chemistry Liver/*chemistry Liver Neoplasms, KEITH Animals Cells, Non-U.S. Gov't Tumor Cells, Transfer, Transfer/*chemistry/isolation & purification RNA, Unité ARN
@article{,
title = {Determination of queuosine derivatives by reverse-phase liquid chromatography for the hypomodification study of Q-bearing tRNAs from various mammal liver cells},
author = {A Costa and J P Pais de Barros and G Keith and W Baranowski and J Desgres},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14751792},
isbn = {14751792},
year = {2004},
date = {2004-01-01},
journal = {J Chromatogr B Analyt Technol Biomed Life Sci},
volume = {801},
number = {2},
pages = {237-247},
abstract = {Three queuosine derivatives (Q-derivatives) have been found at position 34 of four mammalian so-called Q-tRNAs: queuosine (Q) in tRNA(Asn) and tRNA(His), mannosyl-queuosine (manQ) in tRNA(Asp), and galactosyl-queuosine (galQ) in tRNA(Tyr). An analytical procedure based on the combined means of purified tRNA isolation from liver cells and ribonucleoside analysis by reverse-phase high performance liquid chromatography coupled with real-time UV-spectrometry (RPLC-UV) was developed for the quantitative analysis of the three Q-derivatives present in total tRNA from liver tissues and liver cell cultures. Using this analytical procedure, the rates of Q-tRNA modification were studied in total tRNAs from various mammalian hepatic cells. Our results show that the four Q-tRNAs are fully modified in liver tissues from adult mammals, regardless of the mammal species. However, a lack in the Q-modification level was observed in Q-tRNAs from newborn rat liver, as well in Q-tRNAs from normal rat liver cell cultures growing in a low queuine content medium, and from a rat hepatoma cell line. It is noteworthy that in all cases of Q-tRNA hypomodification, our analytical procedure showed that tRNA(Asp) is always the least affected by the hypomodification. The biological significance of this phenomenon is discussed.},
note = {1570-0232
Journal Article},
keywords = {Amino Acyl/chemistry RNA, Asn/chemistry Rats Support, Cultured, Cultured Chickens *Chromatography, Experimental Nucleoside Q/*analogs & derivatives/*analysis RNA, High Pressure Liquid Hepatocytes/chemistry Liver/*chemistry Liver Neoplasms, KEITH Animals Cells, Non-U.S. Gov't Tumor Cells, Transfer, Transfer/*chemistry/isolation & purification RNA, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Three queuosine derivatives (Q-derivatives) have been found at position 34 of four mammalian so-called Q-tRNAs: queuosine (Q) in tRNA(Asn) and tRNA(His), mannosyl-queuosine (manQ) in tRNA(Asp), and galactosyl-queuosine (galQ) in tRNA(Tyr). An analytical procedure based on the combined means of purified tRNA isolation from liver cells and ribonucleoside analysis by reverse-phase high performance liquid chromatography coupled with real-time UV-spectrometry (RPLC-UV) was developed for the quantitative analysis of the three Q-derivatives present in total tRNA from liver tissues and liver cell cultures. Using this analytical procedure, the rates of Q-tRNA modification were studied in total tRNAs from various mammalian hepatic cells. Our results show that the four Q-tRNAs are fully modified in liver tissues from adult mammals, regardless of the mammal species. However, a lack in the Q-modification level was observed in Q-tRNAs from newborn rat liver, as well in Q-tRNAs from normal rat liver cell cultures growing in a low queuine content medium, and from a rat hepatoma cell line. It is noteworthy that in all cases of Q-tRNA hypomodification, our analytical procedure showed that tRNA(Asp) is always the least affected by the hypomodification. The biological significance of this phenomenon is discussed.
Campanacci V, Dubois D Y, Becker H D, Kern D, Spinelli S, Valencia C, Pagot F, Salomoni A, Grisel S, Vincentelli R, Bignon C, Lapointe J, Giege R, Cambillau C
The Escherichia coli YadB gene product reveals a novel aminoacyl-tRNA synthetase like activity Journal Article
In: J Mol Biol, vol. 337, no. 2, pp. 273-283, 2004, ISBN: 15003446, (0022-2836 Journal Article).
Abstract | Links | BibTeX | Tags: Amino Acid Support, Bacterial Glutamate-tRNA Ligase/chemistry/genetics/metabolism Glutamic Acid/metabolism Kinetics Ligands Models, Glu/metabolism Sequence Homology, KERN GIEGE Adenosine Monophosphate/metabolism Adenosine Triphosphate/metabolism Amino Acid Sequence Amino Acyl-tRNA Ligases/chemistry/*genetics/*metabolism Carrier Proteins/metabolism Crystallography, Molecular Molecular Sequence Data Neoplasm Proteins/metabolism Nuclear Proteins/metabolism Protein Conformation RNA, Non-U.S. Gov't Thermus thermophilus/enzymology/genetics Zinc/metabolism, Transfer, Unité ARN, X-Ray Escherichia coli/*enzymology/*genetics Escherichia coli Proteins/chemistry/*genetics/*metabolism Genes
@article{,
title = {The Escherichia coli YadB gene product reveals a novel aminoacyl-tRNA synthetase like activity},
author = {V Campanacci and D Y Dubois and H D Becker and D Kern and S Spinelli and C Valencia and F Pagot and A Salomoni and S Grisel and R Vincentelli and C Bignon and J Lapointe and R Giege and C Cambillau},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15003446},
isbn = {15003446},
year = {2004},
date = {2004-01-01},
journal = {J Mol Biol},
volume = {337},
number = {2},
pages = {273-283},
abstract = {In the course of a structural genomics program aiming at solving the structures of Escherichia coli open reading frame products of unknown function, we have determined the structure of YadB at 1.5A using molecular replacement. The YadB protein is 298 amino acid residues long and displays 34% sequence identity with E.coli glutamyl-tRNA synthetase (GluRS). It is much shorter than GluRS, which contains 468 residues, and lacks the complete domain interacting with the tRNA anticodon loop. As E.coli GluRS, YadB possesses a Zn2+ located in the putative tRNA acceptor stem-binding domain. The YadB cluster uses cysteine residues as the first three zinc ligands, but has a weaker tyrosine ligand at the fourth position. It shares with canonical amino acid RNA synthetases a major functional feature, namely activation of the amino acid (here glutamate). It differs, however, from GluRSs by the fact that the activation step is tRNA-independent and that it does not catalyze attachment of the activated glutamate to E.coli tRNAGlu, but to another, as yet unknown tRNA. These results suggest thus a novel function, distinct from that of GluRSs, for the yadB gene family.},
note = {0022-2836
Journal Article},
keywords = {Amino Acid Support, Bacterial Glutamate-tRNA Ligase/chemistry/genetics/metabolism Glutamic Acid/metabolism Kinetics Ligands Models, Glu/metabolism Sequence Homology, KERN GIEGE Adenosine Monophosphate/metabolism Adenosine Triphosphate/metabolism Amino Acid Sequence Amino Acyl-tRNA Ligases/chemistry/*genetics/*metabolism Carrier Proteins/metabolism Crystallography, Molecular Molecular Sequence Data Neoplasm Proteins/metabolism Nuclear Proteins/metabolism Protein Conformation RNA, Non-U.S. Gov't Thermus thermophilus/enzymology/genetics Zinc/metabolism, Transfer, Unité ARN, X-Ray Escherichia coli/*enzymology/*genetics Escherichia coli Proteins/chemistry/*genetics/*metabolism Genes},
pubstate = {published},
tppubtype = {article}
}
In the course of a structural genomics program aiming at solving the structures of Escherichia coli open reading frame products of unknown function, we have determined the structure of YadB at 1.5A using molecular replacement. The YadB protein is 298 amino acid residues long and displays 34% sequence identity with E.coli glutamyl-tRNA synthetase (GluRS). It is much shorter than GluRS, which contains 468 residues, and lacks the complete domain interacting with the tRNA anticodon loop. As E.coli GluRS, YadB possesses a Zn2+ located in the putative tRNA acceptor stem-binding domain. The YadB cluster uses cysteine residues as the first three zinc ligands, but has a weaker tyrosine ligand at the fourth position. It shares with canonical amino acid RNA synthetases a major functional feature, namely activation of the amino acid (here glutamate). It differs, however, from GluRSs by the fact that the activation step is tRNA-independent and that it does not catalyze attachment of the activated glutamate to E.coli tRNAGlu, but to another, as yet unknown tRNA. These results suggest thus a novel function, distinct from that of GluRSs, for the yadB gene family.
Blaise M, Becker H D, Keith G, Cambillau C, Lapointe J, Giege R, Kern D
A minimalist glutamyl-tRNA synthetase dedicated to aminoacylation of the tRNAAsp QUC anticodon Journal Article
In: Nucleic Acids Res, vol. 32, no. 9, pp. 2768-2775, 2004, ISBN: 15150343, (1362-4962 Journal Article).
Abstract | Links | BibTeX | Tags: Asp/chemistry/genetics/*metabolism RNA, Bacterial/chemistry/genetics/metabolism RNA, Glu/chemistry/genetics/metabolism Support, KERN Acylation Anticodon/chemistry/genetics/*metabolism Base Sequence Conserved Sequence Escherichia coli/*enzymology/*genetics Evolution Glutamate-tRNA Ligase/*chemistry/genetics/*metabolism Molecular Mimicry Nucleoside Q/genetics/*metabolism Periodic Acid/pharmacology RNA, Non-U.S. Gov't, Transfer, Unité ARN
@article{,
title = {A minimalist glutamyl-tRNA synthetase dedicated to aminoacylation of the tRNAAsp QUC anticodon},
author = {M Blaise and H D Becker and G Keith and C Cambillau and J Lapointe and R Giege and D Kern},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15150343},
isbn = {15150343},
year = {2004},
date = {2004-01-01},
journal = {Nucleic Acids Res},
volume = {32},
number = {9},
pages = {2768-2775},
abstract = {Escherichia coli encodes YadB, a protein displaying 34% identity with the catalytic core of glutamyl-tRNA synthetase but lacking the anticodon-binding domain. We show that YadB is a tRNA modifying enzyme that evidently glutamylates the queuosine residue, a modified nucleoside at the wobble position of the tRNA(Asp) QUC anticodon. This conclusion is supported by a variety of biochemical data and by the inability of the enzyme to glutamylate tRNA(Asp) isolated from an E.coli tRNA-guanosine transglycosylase minus strain deprived of the capacity to exchange guanosine 34 with queuosine. Structural mimicry between the tRNA(Asp) anticodon stem and the tRNA(Glu) amino acid acceptor stem in prokaryotes encoding YadB proteins indicates that the function of these tRNA modifying enzymes, which we rename glutamyl-Q tRNA(Asp) synthetases, is conserved among prokaryotes.},
note = {1362-4962
Journal Article},
keywords = {Asp/chemistry/genetics/*metabolism RNA, Bacterial/chemistry/genetics/metabolism RNA, Glu/chemistry/genetics/metabolism Support, KERN Acylation Anticodon/chemistry/genetics/*metabolism Base Sequence Conserved Sequence Escherichia coli/*enzymology/*genetics Evolution Glutamate-tRNA Ligase/*chemistry/genetics/*metabolism Molecular Mimicry Nucleoside Q/genetics/*metabolism Periodic Acid/pharmacology RNA, Non-U.S. Gov't, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Escherichia coli encodes YadB, a protein displaying 34% identity with the catalytic core of glutamyl-tRNA synthetase but lacking the anticodon-binding domain. We show that YadB is a tRNA modifying enzyme that evidently glutamylates the queuosine residue, a modified nucleoside at the wobble position of the tRNA(Asp) QUC anticodon. This conclusion is supported by a variety of biochemical data and by the inability of the enzyme to glutamylate tRNA(Asp) isolated from an E.coli tRNA-guanosine transglycosylase minus strain deprived of the capacity to exchange guanosine 34 with queuosine. Structural mimicry between the tRNA(Asp) anticodon stem and the tRNA(Glu) amino acid acceptor stem in prokaryotes encoding YadB proteins indicates that the function of these tRNA modifying enzymes, which we rename glutamyl-Q tRNA(Asp) synthetases, is conserved among prokaryotes.
Ador L, Jaeger S, Geslain R, Martin F, Cavarelli J, Eriani G
Mutation and evolution of the magnesium-binding site of a class II aminoacyl-tRNA synthetase Journal Article
In: Biochemistry, vol. 43, no. 22, pp. 7028-7037, 2004, ISBN: 15170340, (0006-2960 Journal Article).
Abstract | Links | BibTeX | Tags: Acylation Adenosine Triphosphate/metabolism Amino Acid Substitution Aspartate-tRNA Ligase/chemistry/genetics/*metabolism Aspartic Acid/metabolism Binding Sites Catalytic Domain Cell Death Combinatorial Chemistry Techniques Comparative Study *Evolution, Asp/metabolism Saccharomyces cerevisiae/*enzymology Support, ERIANI, Molecular Kinetics Magnesium/*metabolism Mutagenesis, Non-U.S. Gov't Transfection, Site-Directed Mutation/*genetics Peptide Library Protein Binding Protein Conformation RNA, Transfer, Unité ARN
@article{,
title = {Mutation and evolution of the magnesium-binding site of a class II aminoacyl-tRNA synthetase},
author = {L Ador and S Jaeger and R Geslain and F Martin and J Cavarelli and G Eriani},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15170340},
isbn = {15170340},
year = {2004},
date = {2004-01-01},
journal = {Biochemistry},
volume = {43},
number = {22},
pages = {7028-7037},
abstract = {Aminoacyl-tRNA synthetases contain one or three Mg(2+) ions in their catalytic sites. In addition to their role in ATP binding, these ions are presumed to play a role in catalysis by increasing the electropositivity of the alpha-phosphate and stabilizing the pentavalent transition state. In the class II aaRS, two highly conserved carboxylate residues have been shown to participate with Mg(2+) ions in binding and coordination. It is shown here that these carboxylate residues are absolutely required for the activity of Saccharomyces cerevisiae aspartyl-tRNA synthetase. Mutants of these residues exhibit pleiotropic effects on the kinetic parameters suggesting an effect at an early stage of the aminoacylation reaction, such as the binding of ATP, Mg(2+), aspartic acid, or the amino acid activation. Despite genetic selections in an APS-knockout yeast strain, we were unable to select a single active mutant of these carboxylate residues. Nevertheless, we isolated an intragenic suppressor from a combinatorial library. The active mutant showed a second substitution close to the first one, and exhibited a significant increase of the tRNA aminoacylation rate. Structural analysis suggests that the acceptor stem of the tRNA might be repositioned to give a more productive enzyme:tRNA complex. Thus, the initial defect of the activation reaction was compensated by a significant increase of the aminoacylation rate that led to cellular complementation.},
note = {0006-2960
Journal Article},
keywords = {Acylation Adenosine Triphosphate/metabolism Amino Acid Substitution Aspartate-tRNA Ligase/chemistry/genetics/*metabolism Aspartic Acid/metabolism Binding Sites Catalytic Domain Cell Death Combinatorial Chemistry Techniques Comparative Study *Evolution, Asp/metabolism Saccharomyces cerevisiae/*enzymology Support, ERIANI, Molecular Kinetics Magnesium/*metabolism Mutagenesis, Non-U.S. Gov't Transfection, Site-Directed Mutation/*genetics Peptide Library Protein Binding Protein Conformation RNA, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Aminoacyl-tRNA synthetases contain one or three Mg(2+) ions in their catalytic sites. In addition to their role in ATP binding, these ions are presumed to play a role in catalysis by increasing the electropositivity of the alpha-phosphate and stabilizing the pentavalent transition state. In the class II aaRS, two highly conserved carboxylate residues have been shown to participate with Mg(2+) ions in binding and coordination. It is shown here that these carboxylate residues are absolutely required for the activity of Saccharomyces cerevisiae aspartyl-tRNA synthetase. Mutants of these residues exhibit pleiotropic effects on the kinetic parameters suggesting an effect at an early stage of the aminoacylation reaction, such as the binding of ATP, Mg(2+), aspartic acid, or the amino acid activation. Despite genetic selections in an APS-knockout yeast strain, we were unable to select a single active mutant of these carboxylate residues. Nevertheless, we isolated an intragenic suppressor from a combinatorial library. The active mutant showed a second substitution close to the first one, and exhibited a significant increase of the tRNA aminoacylation rate. Structural analysis suggests that the acceptor stem of the tRNA might be repositioned to give a more productive enzyme:tRNA complex. Thus, the initial defect of the activation reaction was compensated by a significant increase of the aminoacylation rate that led to cellular complementation.
2003
Zhao M W, Hao R, Chen J F, Martin F, Eriani G, Wang E D
Enzymes assembled from Aquifex aeolicus and Escherichia coli leucyl-tRNA synthetases Journal Article
In: Biochemistry, vol. 42, no. 25, pp. 7694-7700, 2003, ISBN: 12820878, (0006-2960 Journal Article).
Abstract | Links | BibTeX | Tags: Amino Acyl-tRNA Ligases/genetics/*metabolism Escherichia coli/*enzymology Evolution, ERIANI, Horizontal Heat Kinetics Mutation RNA, Leu/*metabolism Structure-Activity Relationship Support, Molecular Gene Transfer, Non-U.S. Gov't, Transfer, Unité ARN
@article{,
title = {Enzymes assembled from Aquifex aeolicus and Escherichia coli leucyl-tRNA synthetases},
author = {M W Zhao and R Hao and J F Chen and F Martin and G Eriani and E D Wang},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12820878},
isbn = {12820878},
year = {2003},
date = {2003-01-01},
journal = {Biochemistry},
volume = {42},
number = {25},
pages = {7694-7700},
abstract = {Aquifex aeolicus alphabeta-LeuRS is the only known heterodimeric LeuRS, while Escherichia coli LeuRS is a canonical monomeric enzyme. By using the genes encoding A. aeolicus and E. coli LeuRS as PCR templates, the genes encoding the alpha and beta subunits from A. aeolicus alphabeta-LeuRS and the equivalent amino- and carboxy-terminal parts of E. coli LeuRS (identified as alpha' and beta') were amplified and recombined using suitable plasmids. These recombinant plasmids were transformed or cotransformed into E. coli to produce five monomeric and five heterodimeric LeuRS mutants. Seven of these were successfully overexpressed in vivo and purified, while three dimeric mutants with the beta' part of E. coli LeuRS were not successfully expressed. The seven purified mutants catalyzed amino acid activation, although several exhibited reduced aminoacylation properties. Removal of the last 36 residues of the alpha subunit of the A. aeolicus enzyme was determined to be deleterious for tRNA charging. Indeed, subunit exchange showed that the cross-species-specific recognition of A. aeolicus tRNA(Leu) occurs at the alpha subunit. None of the mixed E. coli-A. aeolicus enzymes were as thermostable as the native alphabeta-LeuRS. However, the fusion of the two alpha and beta peptides from A. aeolicus as a single chain analogous to canonical LeuRS resulted in a product more resistant to heat denaturation than the original enzyme.},
note = {0006-2960
Journal Article},
keywords = {Amino Acyl-tRNA Ligases/genetics/*metabolism Escherichia coli/*enzymology Evolution, ERIANI, Horizontal Heat Kinetics Mutation RNA, Leu/*metabolism Structure-Activity Relationship Support, Molecular Gene Transfer, Non-U.S. Gov't, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Aquifex aeolicus alphabeta-LeuRS is the only known heterodimeric LeuRS, while Escherichia coli LeuRS is a canonical monomeric enzyme. By using the genes encoding A. aeolicus and E. coli LeuRS as PCR templates, the genes encoding the alpha and beta subunits from A. aeolicus alphabeta-LeuRS and the equivalent amino- and carboxy-terminal parts of E. coli LeuRS (identified as alpha' and beta') were amplified and recombined using suitable plasmids. These recombinant plasmids were transformed or cotransformed into E. coli to produce five monomeric and five heterodimeric LeuRS mutants. Seven of these were successfully overexpressed in vivo and purified, while three dimeric mutants with the beta' part of E. coli LeuRS were not successfully expressed. The seven purified mutants catalyzed amino acid activation, although several exhibited reduced aminoacylation properties. Removal of the last 36 residues of the alpha subunit of the A. aeolicus enzyme was determined to be deleterious for tRNA charging. Indeed, subunit exchange showed that the cross-species-specific recognition of A. aeolicus tRNA(Leu) occurs at the alpha subunit. None of the mixed E. coli-A. aeolicus enzymes were as thermostable as the native alphabeta-LeuRS. However, the fusion of the two alpha and beta peptides from A. aeolicus as a single chain analogous to canonical LeuRS resulted in a product more resistant to heat denaturation than the original enzyme.
Sohm B, Frugier M, Brule H, Olszak K, Przykorska A, Florentz C
Towards understanding human mitochondrial leucine aminoacylation identity Journal Article
In: J Mol Biol, vol. 328, no. 5, pp. 995-1010, 2003, ISBN: 12729737, (0022-2836 Journal Article).
Abstract | Links | BibTeX | Tags: Base Sequence Human In Vitro Leucine/*metabolism Leucine-tRNA Ligase/*metabolism Mitochondria/*metabolism Mitochondrial Diseases/genetics/metabolism Molecular Sequence Data Mutation Nucleic Acid Conformation RNA, ERIANI, FLORENTZ, FRUGIER, Leu/chemistry/genetics/*metabolism Recombinant Proteins/genetics/metabolism Solutions Substrate Specificity Support, Non-U.S. Gov't Variation (Genetics), Transfer, Unité ARN
@article{,
title = {Towards understanding human mitochondrial leucine aminoacylation identity},
author = {B Sohm and M Frugier and H Brule and K Olszak and A Przykorska and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12729737},
isbn = {12729737},
year = {2003},
date = {2003-01-01},
journal = {J Mol Biol},
volume = {328},
number = {5},
pages = {995-1010},
abstract = {Specific recognition of tRNAs by aminoacyl-tRNA synthetases is governed by sets of aminoacylation identity elements, well defined for numerous prokaryotic systems and eukaryotic cytosolic systems. Only restricted information is available for aminoacylation of human mitochondrial tRNAs, despite their particularities linked to the non-classical structures of the tRNAs and their involvement in a growing number of human neurodegenerative disorders linked to mutations in the corresponding tRNA genes. A major difficulty to be overcome is the preparation of active in vitro transcripts enabling a rational mutagenic analysis, as is currently performed for classical tRNAs. Here, structural and aminoacylation properties of in vitro transcribed tRNA(Leu(UUR)) are presented. Solution probing using a combination of enzymatic and chemical tools revealed only partial folding into an L-shaped structure, with an acceptor branch but with a floppy anticodon branch. Optimization of aminoacylation conditions allowed charging of up to 75% of molecules, showing that, despite its partially relaxed structure, in vitro transcribed tRNA(Leu(UUR)) is able to adapt to the synthetase. In addition, mutational analysis demonstrates that the discriminator base as well as residue A14 are important leucine identity elements. Thus, human mitochondrial leucylation is dependent on rules similar to those that apply in Escherichia coli. The impact of a subset of pathology-related mutations on aminoacylation and on tRNA structure, has been explored. These variants do not show significant structural rearrangements and either do not affect aminoacylation (mutations T3250C, T3271C, C3303T) or lead to marked effects. Interestingly, two variants with a mutation at the same position (A3243G and A3243T) lead to markedly different losses in aminoacylation efficiencies (tenfold and 300-fold, respectively).},
note = {0022-2836
Journal Article},
keywords = {Base Sequence Human In Vitro Leucine/*metabolism Leucine-tRNA Ligase/*metabolism Mitochondria/*metabolism Mitochondrial Diseases/genetics/metabolism Molecular Sequence Data Mutation Nucleic Acid Conformation RNA, ERIANI, FLORENTZ, FRUGIER, Leu/chemistry/genetics/*metabolism Recombinant Proteins/genetics/metabolism Solutions Substrate Specificity Support, Non-U.S. Gov't Variation (Genetics), Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Specific recognition of tRNAs by aminoacyl-tRNA synthetases is governed by sets of aminoacylation identity elements, well defined for numerous prokaryotic systems and eukaryotic cytosolic systems. Only restricted information is available for aminoacylation of human mitochondrial tRNAs, despite their particularities linked to the non-classical structures of the tRNAs and their involvement in a growing number of human neurodegenerative disorders linked to mutations in the corresponding tRNA genes. A major difficulty to be overcome is the preparation of active in vitro transcripts enabling a rational mutagenic analysis, as is currently performed for classical tRNAs. Here, structural and aminoacylation properties of in vitro transcribed tRNA(Leu(UUR)) are presented. Solution probing using a combination of enzymatic and chemical tools revealed only partial folding into an L-shaped structure, with an acceptor branch but with a floppy anticodon branch. Optimization of aminoacylation conditions allowed charging of up to 75% of molecules, showing that, despite its partially relaxed structure, in vitro transcribed tRNA(Leu(UUR)) is able to adapt to the synthetase. In addition, mutational analysis demonstrates that the discriminator base as well as residue A14 are important leucine identity elements. Thus, human mitochondrial leucylation is dependent on rules similar to those that apply in Escherichia coli. The impact of a subset of pathology-related mutations on aminoacylation and on tRNA structure, has been explored. These variants do not show significant structural rearrangements and either do not affect aminoacylation (mutations T3250C, T3271C, C3303T) or lead to marked effects. Interestingly, two variants with a mutation at the same position (A3243G and A3243T) lead to markedly different losses in aminoacylation efficiencies (tenfold and 300-fold, respectively).
Ryckelynck M, Giege R, Frugier M
Yeast tRNA(Asp) charging accuracy is threatened by the N-terminal extension of aspartyl-tRNA synthetase Journal Article
In: J Biol Chem, vol. 278, no. 11, pp. 9683-9690, 2003, ISBN: 12486031, (0021-9258 Journal Article).
Abstract | Links | BibTeX | Tags: Amino Acid Motifs Aspartate-tRNA Ligase/*metabolism Aspartic Acid/chemistry Base Sequence Codon Escherichia coli/metabolism Kinetics Molecular Sequence Data Nucleic Acid Conformation Nucleic Acids/chemistry Protein Structure, Asp/*chemistry Support, FRUGIER, Messenger/metabolism RNA, Non-U.S. Gov't Yeasts/metabolism, Secondary Protein Structure, Tertiary RNA, Transfer, Unité ARN
@article{,
title = {Yeast tRNA(Asp) charging accuracy is threatened by the N-terminal extension of aspartyl-tRNA synthetase},
author = {M Ryckelynck and R Giege and M Frugier},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12486031},
isbn = {12486031},
year = {2003},
date = {2003-01-01},
journal = {J Biol Chem},
volume = {278},
number = {11},
pages = {9683-9690},
abstract = {This study evaluates the role of the N-terminal extension from yeast aspartyl-tRNA synthetase in tRNA aspartylation. The presence of an RNA-binding motif in this extension, conserved in eukaryotic class IIb aminoacyl-tRNA synthetases, provides nonspecific tRNA binding properties to this enzyme. Here, it is assumed that the additional contacts the 70 amino acid-long appendix of aspartyl-tRNA synthetase makes with tRNA could be important in expression of aspartate identity in yeast. Using in vitro transcripts mutated at identity positions, it is demonstrated that the extension grants better aminoacylation efficiency but reduced specificity to the synthetase, increasing considerably the risk of noncognate tRNA mischarging. Yeast tRNA(Glu(UUC)) and tRNA(Asn(GUU)) were identified as the most easily mischarged tRNA species. Both have a G at the discriminator position, and their anticodon differs only by one change from the GUC aspartate anticodon.},
note = {0021-9258
Journal Article},
keywords = {Amino Acid Motifs Aspartate-tRNA Ligase/*metabolism Aspartic Acid/chemistry Base Sequence Codon Escherichia coli/metabolism Kinetics Molecular Sequence Data Nucleic Acid Conformation Nucleic Acids/chemistry Protein Structure, Asp/*chemistry Support, FRUGIER, Messenger/metabolism RNA, Non-U.S. Gov't Yeasts/metabolism, Secondary Protein Structure, Tertiary RNA, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
This study evaluates the role of the N-terminal extension from yeast aspartyl-tRNA synthetase in tRNA aspartylation. The presence of an RNA-binding motif in this extension, conserved in eukaryotic class IIb aminoacyl-tRNA synthetases, provides nonspecific tRNA binding properties to this enzyme. Here, it is assumed that the additional contacts the 70 amino acid-long appendix of aspartyl-tRNA synthetase makes with tRNA could be important in expression of aspartate identity in yeast. Using in vitro transcripts mutated at identity positions, it is demonstrated that the extension grants better aminoacylation efficiency but reduced specificity to the synthetase, increasing considerably the risk of noncognate tRNA mischarging. Yeast tRNA(Glu(UUC)) and tRNA(Asn(GUU)) were identified as the most easily mischarged tRNA species. Both have a G at the discriminator position, and their anticodon differs only by one change from the GUC aspartate anticodon.
Romby P, Springer M
Bacterial translational control at atomic resolution Journal Article
In: Trends Genet, vol. 19, no. 3, pp. 155-161, 2003, ISBN: 12615010, (0168-9525 Journal Article Review Review, Tutorial).
Abstract | Links | BibTeX | Tags: Bacterial *Gene Expression Regulation, Bacterial Models, Base Sequence Conserved Sequence Escherichia coli/*enzymology/*genetics *Gene Expression Regulation, Biological Models, Enzymologic Genes, Genetic, Messenger/genetics/*metabolism RNA, Molecular Molecular Mimicry Nucleic Acid Conformation Operator Regions (Genetics) RNA, Non-U.S. Gov't Threonine-tRNA Ligase/chemistry/*genetics/metabolism *Translation, ROMBY, Thr/*metabolism RNA-Binding Proteins/metabolism Support, Transfer, Unité ARN
@article{,
title = {Bacterial translational control at atomic resolution},
author = {P Romby and M Springer},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12615010},
isbn = {12615010},
year = {2003},
date = {2003-01-01},
journal = {Trends Genet},
volume = {19},
number = {3},
pages = {155-161},
abstract = {Translational regulation allows rapid adaptation of protein synthesis to environmental conditions. In prokaryotes, the synthesis of many RNA-binding proteins is regulated by a translational feedback mechanism involving a competition between their natural substrate and their binding site on mRNA, which are often thought to resemble each other. This article describes the case of threonyl-tRNA synthetase, which represses the translation of its own mRNA. Recent data provide the first opportunity to describe at the atomic level both the extent and the limit of mimicry between the way this enzyme recognizes tRNA(Thr) and its regulatory site in mRNA. The data also give some clues about how the binding of the synthetase to its mRNA inhibits translation.},
note = {0168-9525
Journal Article
Review
Review, Tutorial},
keywords = {Bacterial *Gene Expression Regulation, Bacterial Models, Base Sequence Conserved Sequence Escherichia coli/*enzymology/*genetics *Gene Expression Regulation, Biological Models, Enzymologic Genes, Genetic, Messenger/genetics/*metabolism RNA, Molecular Molecular Mimicry Nucleic Acid Conformation Operator Regions (Genetics) RNA, Non-U.S. Gov't Threonine-tRNA Ligase/chemistry/*genetics/metabolism *Translation, ROMBY, Thr/*metabolism RNA-Binding Proteins/metabolism Support, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Translational regulation allows rapid adaptation of protein synthesis to environmental conditions. In prokaryotes, the synthesis of many RNA-binding proteins is regulated by a translational feedback mechanism involving a competition between their natural substrate and their binding site on mRNA, which are often thought to resemble each other. This article describes the case of threonyl-tRNA synthetase, which represses the translation of its own mRNA. Recent data provide the first opportunity to describe at the atomic level both the extent and the limit of mimicry between the way this enzyme recognizes tRNA(Thr) and its regulatory site in mRNA. The data also give some clues about how the binding of the synthetase to its mRNA inhibits translation.
Rigourd M, Goldschmidt V, Brule F, Morrow C D, Ehresmann B, Ehresmann C, Marquet R
Structure-function relationships of the initiation complex of HIV-1 reverse transcription: the case of mutant viruses using tRNA(His) as primer Journal Article
In: Nucleic Acids Res, vol. 31, no. 19, pp. 5764-5775, 2003, ISBN: 14500840, (1362-4962 Journal Article).
Abstract | Links | BibTeX | Tags: Base Sequence Comparative Study DNA, Genetic, Genetic *Transcription Initiation Site *Transcription, His/*metabolism RNA, MARQUET, Non-U.S. Gov't Templates, Post-Transcriptional RNA, Transfer, Unité ARN, Viral HIV-1/*genetics/metabolism HIV-1 Reverse Transcriptase/metabolism Kinetics Macromolecular Systems Molecular Sequence Data Mutation RNA Probes RNA Processing, Viral/*biosynthesis/genetics Sequence Alignment Structure-Activity Relationship Support, Viral/biosynthesis *Gene Expression Regulation
@article{,
title = {Structure-function relationships of the initiation complex of HIV-1 reverse transcription: the case of mutant viruses using tRNA(His) as primer},
author = {M Rigourd and V Goldschmidt and F Brule and C D Morrow and B Ehresmann and C Ehresmann and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14500840},
isbn = {14500840},
year = {2003},
date = {2003-01-01},
journal = {Nucleic Acids Res},
volume = {31},
number = {19},
pages = {5764-5775},
abstract = {Reverse transcription of HIV-1 RNA is initiated from the 3' end of a tRNA3Lys molecule annealed to the primer binding site (PBS). An additional interaction between the anticodon loop of tRNA3Lys and a viral A-rich loop is required for efficient initiation of reverse transcription of the HIV-1 MAL isolate. In the HIV-1 HXB2 isolate, simultaneous mutations of the PBS and the A-rich loop (mutant His-AC), but not of the PBS alone (mutant His) allows the virus to stably utilize tRNA(His) as primer. However, mutant His-AC selects additional mutations during cell culture, generating successively His-AC-GAC and His-AC-AT-GAC. Here, we wanted to establish direct relationships between the evolution of these mutants in cell culture, their efficiency in initiating reverse transcription and the structure of the primer/template complexes in vitro. The initiation of reverse transcription of His and His-AC RNAs was dramatically reduced. However, His-AC-GAC RNA, which incorporated three adaptative point mutations, was reverse transcribed more efficiently than the wild type RNA. Incorporation of two additional mutations decreased the efficiency of the initiation of reverse transcription, which remained at the wild type level. Structural probing showed that even though both His-AC and His-AC-GAC RNAs can potentially interact with the anticodon loop of tRNA(His), only the latter template formed a stable interaction. Thus, our results showed that the selection of adaptative mutations by HIV-1 mutants utilizing tRNA(His) as primer was initially dictated by the efficiency of the initiation of reverse transcription, which relied on the existence of a stable interaction between the mutated A-rich loop and the anticodon loop of tRNA(His).},
note = {1362-4962
Journal Article},
keywords = {Base Sequence Comparative Study DNA, Genetic, Genetic *Transcription Initiation Site *Transcription, His/*metabolism RNA, MARQUET, Non-U.S. Gov't Templates, Post-Transcriptional RNA, Transfer, Unité ARN, Viral HIV-1/*genetics/metabolism HIV-1 Reverse Transcriptase/metabolism Kinetics Macromolecular Systems Molecular Sequence Data Mutation RNA Probes RNA Processing, Viral/*biosynthesis/genetics Sequence Alignment Structure-Activity Relationship Support, Viral/biosynthesis *Gene Expression Regulation},
pubstate = {published},
tppubtype = {article}
}
Reverse transcription of HIV-1 RNA is initiated from the 3' end of a tRNA3Lys molecule annealed to the primer binding site (PBS). An additional interaction between the anticodon loop of tRNA3Lys and a viral A-rich loop is required for efficient initiation of reverse transcription of the HIV-1 MAL isolate. In the HIV-1 HXB2 isolate, simultaneous mutations of the PBS and the A-rich loop (mutant His-AC), but not of the PBS alone (mutant His) allows the virus to stably utilize tRNA(His) as primer. However, mutant His-AC selects additional mutations during cell culture, generating successively His-AC-GAC and His-AC-AT-GAC. Here, we wanted to establish direct relationships between the evolution of these mutants in cell culture, their efficiency in initiating reverse transcription and the structure of the primer/template complexes in vitro. The initiation of reverse transcription of His and His-AC RNAs was dramatically reduced. However, His-AC-GAC RNA, which incorporated three adaptative point mutations, was reverse transcribed more efficiently than the wild type RNA. Incorporation of two additional mutations decreased the efficiency of the initiation of reverse transcription, which remained at the wild type level. Structural probing showed that even though both His-AC and His-AC-GAC RNAs can potentially interact with the anticodon loop of tRNA(His), only the latter template formed a stable interaction. Thus, our results showed that the selection of adaptative mutations by HIV-1 mutants utilizing tRNA(His) as primer was initially dictated by the efficiency of the initiation of reverse transcription, which relied on the existence of a stable interac