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Modèles Insectes d’Immunité Innée
Publications
2008
Geary C., Baudrey S., Jaeger L.
Comprehensive features of natural and in vitro selected GNRA tetraloop-binding receptors Article de journal
Dans: Nucleic Acids Res, vol. 36, non 4, p. 1138-52, 2008, (1362-4962 (Electronic) Journal Article Research Support, N.I.H., Extramural Research Support, U.S. Gov't, Non-P.H.S.).
Résumé | BibTeX | Étiquettes: Acid, Adenine/chemistry, Analysis, Base, Conformation, Data, dimerization, directed, Evolution, KROL, Models, Molecular, Nucleic, RNA, RNA/*chemistry/classification, Sequence, Thermodynamics
@article{,
title = {Comprehensive features of natural and in vitro selected GNRA tetraloop-binding receptors},
author = { C. Geary and S. Baudrey and L. Jaeger},
year = {2008},
date = {2008-01-01},
journal = {Nucleic Acids Res},
volume = {36},
number = {4},
pages = {1138-52},
abstract = {Specific recognitions of GNRA tetraloops by small helical receptors are among the most widespread long-range packing interactions in large ribozymes. However, in contrast to GYRA and GAAA tetraloops, very few GNRA/receptor interactions have yet been identified to involve GGAA tetraloops in nature. A novel in vitro selection scheme based on a rigid self-assembling tectoRNA scaffold designed for isolation of intermolecular interactions with A-minor motifs has yielded new GGAA tetraloop-binding receptors with affinity in the nanomolar range. One of the selected receptors is a novel 12 nt RNA motif, (CCUGUG. AUCUGG), that recognizes GGAA tetraloop hairpin with a remarkable specificity and affinity. Its physical and chemical characteristics are comparable to those of the well-studied '11nt' GAAA tetraloop receptor motif. A second less specific motif (CCCAGCCC. GAUAGGG) binds GGRA tetraloops and appears to be related to group IC3 tetraloop receptors. Mutational, thermodynamic and comparative structural analysis suggests that natural and in vitro selected GNRA receptors can essentially be grouped in two major classes of GNRA binders. New insights about the evolution, recognition and structural modularity of GNRA and A-minor RNA-RNA interactions are proposed.},
note = {1362-4962 (Electronic)
Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.},
keywords = {Acid, Adenine/chemistry, Analysis, Base, Conformation, Data, dimerization, directed, Evolution, KROL, Models, Molecular, Nucleic, RNA, RNA/*chemistry/classification, Sequence, Thermodynamics},
pubstate = {published},
tppubtype = {article}
}
Specific recognitions of GNRA tetraloops by small helical receptors are among the most widespread long-range packing interactions in large ribozymes. However, in contrast to GYRA and GAAA tetraloops, very few GNRA/receptor interactions have yet been identified to involve GGAA tetraloops in nature. A novel in vitro selection scheme based on a rigid self-assembling tectoRNA scaffold designed for isolation of intermolecular interactions with A-minor motifs has yielded new GGAA tetraloop-binding receptors with affinity in the nanomolar range. One of the selected receptors is a novel 12 nt RNA motif, (CCUGUG. AUCUGG), that recognizes GGAA tetraloop hairpin with a remarkable specificity and affinity. Its physical and chemical characteristics are comparable to those of the well-studied '11nt' GAAA tetraloop receptor motif. A second less specific motif (CCCAGCCC. GAUAGGG) binds GGRA tetraloops and appears to be related to group IC3 tetraloop receptors. Mutational, thermodynamic and comparative structural analysis suggests that natural and in vitro selected GNRA receptors can essentially be grouped in two major classes of GNRA binders. New insights about the evolution, recognition and structural modularity of GNRA and A-minor RNA-RNA interactions are proposed.
2004
Grosjean H., Keith G., Droogmans L.
Detection and quantification of modified nucleotides in RNA using thin-layer chromatography Book Section
Dans: Gott, J. M. (Ed.): RNA Interference, Editing, and Modification: Methods and Protocols, vol. 265, p. 357-91, Springer Protocols, Humana Press, 2004.
Résumé | BibTeX | Étiquettes: &, 5', acids, and, Base, Chromatography, Composition, Deoxyribonucleotides/chemistry/isolation, DNA/chemistry/genetics/isolation, Endoribonucleases, Gov't, Indicators, Isotope, KEITH, Labeling/methods, Layer/methods, Non-U.S., Nucleic, Oligoribonucleotides/chemistry/isolation, Peptide, purification, Radioisotopes, Reagents, Regions/chemistry, Ribonucleotides/*analysis, RNA/*genetics/isolation, Support, Thin, Untranslated
@incollection{,
title = {Detection and quantification of modified nucleotides in RNA using thin-layer chromatography},
author = { H. Grosjean and G. Keith and L. Droogmans},
editor = { J.M. Gott},
year = {2004},
date = {2004-01-01},
booktitle = {RNA Interference, Editing, and Modification: Methods and Protocols},
volume = {265},
pages = {357-91},
publisher = {Springer Protocols, Humana Press},
series = {Methods in Molecular Biology},
abstract = {Identification of a modified nucleotide and its localization within an RNA molecule is a difficult task. Only direct sequencing of purified RNA molecules and high-performance liquid chromatography mass spectrometry analysis of purified RNA fragments allow determination of both the type and location of a given modified nucleotide within an RNA of 50-150 nt in length. The objective of this chapter is to describe in detail a few simple procedures that we have found particularly suited for the detection, localization, and quantification of modified nucleotides within an RNA of known sequence. The methods can also be used to reveal the enzymatic activity of a particular RNA-modifying enzyme in vitro or in vivo. The procedures are based on the use of radiolabeled RNA (with [32P], [14C], or [3H]) or [32P]-postlabeled oligonucleotides and two-dimensional thin-layer chromatography of labeled nucleotides on cellulose plates. This chapter provides useful maps of the migration characteristics of 70 modified nucleotides on thin-layer cellulose plates.},
keywords = {&, 5', acids, and, Base, Chromatography, Composition, Deoxyribonucleotides/chemistry/isolation, DNA/chemistry/genetics/isolation, Endoribonucleases, Gov't, Indicators, Isotope, KEITH, Labeling/methods, Layer/methods, Non-U.S., Nucleic, Oligoribonucleotides/chemistry/isolation, Peptide, purification, Radioisotopes, Reagents, Regions/chemistry, Ribonucleotides/*analysis, RNA/*genetics/isolation, Support, Thin, Untranslated},
pubstate = {published},
tppubtype = {incollection}
}
Identification of a modified nucleotide and its localization within an RNA molecule is a difficult task. Only direct sequencing of purified RNA molecules and high-performance liquid chromatography mass spectrometry analysis of purified RNA fragments allow determination of both the type and location of a given modified nucleotide within an RNA of 50-150 nt in length. The objective of this chapter is to describe in detail a few simple procedures that we have found particularly suited for the detection, localization, and quantification of modified nucleotides within an RNA of known sequence. The methods can also be used to reveal the enzymatic activity of a particular RNA-modifying enzyme in vitro or in vivo. The procedures are based on the use of radiolabeled RNA (with [32P], [14C], or [3H]) or [32P]-postlabeled oligonucleotides and two-dimensional thin-layer chromatography of labeled nucleotides on cellulose plates. This chapter provides useful maps of the migration characteristics of 70 modified nucleotides on thin-layer cellulose plates.
Przykorska A., Solecka K., Olszak K., Keith G., Nawrot B., Kuligowska E.
Wheat (Triticum vulgare) chloroplast nuclease ChSI exhibits 5' flap structure-specific endonuclease activity Article de journal
Dans: Biochemistry, vol. 43, non 35, p. 11283-94, 2004, (0006-2960 Journal Article).
Résumé | BibTeX | Étiquettes: &, Acid, Catalysis, Chloroplasts/*enzymology, Conformation, Desorption-Ionization, DNA, Endonucleases/*chemistry/isolation, Exonucleases/chemistry/metabolism, Flap, Gov't, Hydrolysis, KEITH, Kinetics, Laser, Mass, Matrix-Assisted, Non-U.S., Nucleic, Oligonucleotides/chemical, Plant/chemistry/metabolism, purification/*metabolism, Relationship, Single-Stranded/chemistry/metabolism, Specificity, Spectrometry, Structure-Activity, Substrate, Support, synthesis/metabolism, Thermodynamics, Triticum/*enzymology
@article{,
title = {Wheat (Triticum vulgare) chloroplast nuclease ChSI exhibits 5' flap structure-specific endonuclease activity},
author = { A. Przykorska and K. Solecka and K. Olszak and G. Keith and B. Nawrot and E. Kuligowska},
year = {2004},
date = {2004-01-01},
journal = {Biochemistry},
volume = {43},
number = {35},
pages = {11283-94},
abstract = {The structure-specific ChSI nuclease from wheat (Triticum vulgare) chloroplast stroma has been previously purified and characterized in our laboratory. It is a single-strand-specific DNA and RNA endonuclease. Although the enzyme has been initially characterized and used as a structural probe, its biological function is still unknown. Localization of the ChSI enzyme inside chloroplasts, possessing their own DNA that is generally highly exposed to UV light and often affected by numerous redox reactions and electron transfer processes, might suggest, however, that this enzyme could be involved in DNA repair. The repair of some types of DNA damage has been shown to proceed through branched DNA intermediates which are substrates for the structure-specific DNA endonucleases. Thus we tested the substrate specificity of ChSI endonuclease toward various branched DNAs containing 5' flap, 5' pseudoflap, 3' pseudoflap, or single-stranded bulged structural motifs. It appears that ChSI has a high 5' flap structure-specific endonucleolytic activity. The catalytic efficiency (k(cat)/K(M)) of the enzyme is significantly higher for the 5' flap substrate than for single-stranded DNA. The ChSI 5' flap activity was inhibited by high concentrations of Mg(2+), Mn(2+), Zn(2+), or Ca(2+). However, low concentrations of divalent cations could restore the loss of ChSI activity as a consequence of EDTA pretreatment. In contrast to other known 5' flap nucleases, the chloroplast enzyme ChSI does not possess any 5'-->3' exonuclease activity on double-stranded DNA. Therefore, we conclude that ChSI is a 5' flap structure-specific endonuclease with nucleolytic activity toward single-stranded substrates.},
note = {0006-2960
Journal Article},
keywords = {&, Acid, Catalysis, Chloroplasts/*enzymology, Conformation, Desorption-Ionization, DNA, Endonucleases/*chemistry/isolation, Exonucleases/chemistry/metabolism, Flap, Gov't, Hydrolysis, KEITH, Kinetics, Laser, Mass, Matrix-Assisted, Non-U.S., Nucleic, Oligonucleotides/chemical, Plant/chemistry/metabolism, purification/*metabolism, Relationship, Single-Stranded/chemistry/metabolism, Specificity, Spectrometry, Structure-Activity, Substrate, Support, synthesis/metabolism, Thermodynamics, Triticum/*enzymology},
pubstate = {published},
tppubtype = {article}
}
The structure-specific ChSI nuclease from wheat (Triticum vulgare) chloroplast stroma has been previously purified and characterized in our laboratory. It is a single-strand-specific DNA and RNA endonuclease. Although the enzyme has been initially characterized and used as a structural probe, its biological function is still unknown. Localization of the ChSI enzyme inside chloroplasts, possessing their own DNA that is generally highly exposed to UV light and often affected by numerous redox reactions and electron transfer processes, might suggest, however, that this enzyme could be involved in DNA repair. The repair of some types of DNA damage has been shown to proceed through branched DNA intermediates which are substrates for the structure-specific DNA endonucleases. Thus we tested the substrate specificity of ChSI endonuclease toward various branched DNAs containing 5' flap, 5' pseudoflap, 3' pseudoflap, or single-stranded bulged structural motifs. It appears that ChSI has a high 5' flap structure-specific endonucleolytic activity. The catalytic efficiency (k(cat)/K(M)) of the enzyme is significantly higher for the 5' flap substrate than for single-stranded DNA. The ChSI 5' flap activity was inhibited by high concentrations of Mg(2+), Mn(2+), Zn(2+), or Ca(2+). However, low concentrations of divalent cations could restore the loss of ChSI activity as a consequence of EDTA pretreatment. In contrast to other known 5' flap nucleases, the chloroplast enzyme ChSI does not possess any 5'-->3' exonuclease activity on double-stranded DNA. Therefore, we conclude that ChSI is a 5' flap structure-specific endonuclease with nucleolytic activity toward single-stranded substrates.
2003
Bonnal S., Schaeffer C., Creancier L., Clamens S., Moine H., Prats A. C., Vagner S.
A single internal ribosome entry site containing a G quartet RNA structure drives fibroblast growth factor 2 gene expression at four alternative translation initiation codons Article de journal
Dans: J Biol Chem, vol. 278, non 41, p. 39330-6, 2003, (0021-9258 Journal Article).
Résumé | BibTeX | Étiquettes: 2/*genetics, Acid, Alternative, Base, Cell, Chain, Codon, Complementary/genetics, Conformation, Data, Deletion, DNA, Expression, Factor, Fibroblast, Gene, Gov't, Growth, Human, initiation, Initiator/genetics, Line, Messenger/*chemistry/*genetics, Molecular, Non-U.S., Nucleic, Peptide, Ribosomes/*metabolism, RNA, Sequence, Splicing, Support, Transfection
@article{,
title = {A single internal ribosome entry site containing a G quartet RNA structure drives fibroblast growth factor 2 gene expression at four alternative translation initiation codons},
author = { S. Bonnal and C. Schaeffer and L. Creancier and S. Clamens and H. Moine and A. C. Prats and S. Vagner},
year = {2003},
date = {2003-01-01},
journal = {J Biol Chem},
volume = {278},
number = {41},
pages = {39330-6},
abstract = {The 484-nucleotide (nt) alternatively translated region (ATR) of the human fibroblast growth factor 2 (FGF-2) mRNA contains four CUG and one AUG translation initiation codons. Although the 5'-end proximal CUG codon is initiated by a cap-dependent translation process, the other four initiation codons are initiated by a mechanism of internal entry of ribosomes. We undertook here a detailed analysis of the cis-acting elements defining the FGF-2 internal ribosome entry site (IRES). A thorough deletion analysis study within the 5'-ATR led us to define a 176-nt region as being necessary and sufficient for IRES function at four codons present in a downstream 308-nt RNA segment. Unexpectedly, a single IRES module is therefore responsible for translation initiation at four distantly localized codons. The determination of the FGF-2 5'-ATR RNA secondary structure by enzymatic and chemical probing experiments showed that the FGF-2 IRES contained two stem-loop regions and a G quartet motif that constitute novel structural determinants of IRES function.},
note = {0021-9258
Journal Article},
keywords = {2/*genetics, Acid, Alternative, Base, Cell, Chain, Codon, Complementary/genetics, Conformation, Data, Deletion, DNA, Expression, Factor, Fibroblast, Gene, Gov't, Growth, Human, initiation, Initiator/genetics, Line, Messenger/*chemistry/*genetics, Molecular, Non-U.S., Nucleic, Peptide, Ribosomes/*metabolism, RNA, Sequence, Splicing, Support, Transfection},
pubstate = {published},
tppubtype = {article}
}
The 484-nucleotide (nt) alternatively translated region (ATR) of the human fibroblast growth factor 2 (FGF-2) mRNA contains four CUG and one AUG translation initiation codons. Although the 5'-end proximal CUG codon is initiated by a cap-dependent translation process, the other four initiation codons are initiated by a mechanism of internal entry of ribosomes. We undertook here a detailed analysis of the cis-acting elements defining the FGF-2 internal ribosome entry site (IRES). A thorough deletion analysis study within the 5'-ATR led us to define a 176-nt region as being necessary and sufficient for IRES function at four codons present in a downstream 308-nt RNA segment. Unexpectedly, a single IRES module is therefore responsible for translation initiation at four distantly localized codons. The determination of the FGF-2 5'-ATR RNA secondary structure by enzymatic and chemical probing experiments showed that the FGF-2 IRES contained two stem-loop regions and a G quartet motif that constitute novel structural determinants of IRES function.
2002
Cristofari G., Bampi C., Wilhelm M., Wilhelm F. X., Darlix J. L.
A 5'-3' long-range interaction in Ty1 RNA controls its reverse transcription and retrotransposition Article de journal
Dans: EMBO J, vol. 21, non 16, p. 4368-79, 2002, (0261-4189 Journal Article).
Résumé | BibTeX | Étiquettes: *Gene, *Transcription, Acid, cerevisiae/*genetics, Complementary/biosynthesis, Conformation, DNA, Expression, Fungal, Fungal/chemistry/*metabolism, Genetic, Gov't, in, Messenger/chemistry/*metabolism, Non-U.S., Nucleic, Phylogeny, Regulation, Retroelements/*genetics, RNA, Saccharomyces, Support, vitro
@article{,
title = {A 5'-3' long-range interaction in Ty1 RNA controls its reverse transcription and retrotransposition},
author = { G. Cristofari and C. Bampi and M. Wilhelm and F. X. Wilhelm and J. L. Darlix},
year = {2002},
date = {2002-01-01},
journal = {EMBO J},
volume = {21},
number = {16},
pages = {4368-79},
abstract = {LTR-retrotransposons are abundant components of all eukaryotic genomes and appear to be key players in their evolution. They share with retroviruses a reverse transcription step during their replication cycle. To better understand the replication of retrotransposons as well as their similarities to and differences from retroviruses, we set up an in vitro model system to examine minus-strand cDNA synthesis of the yeast Ty1 LTR-retrotransposon. Results show that the 5' and 3' ends of Ty1 genomic RNA interact through 14 nucleotide 5'-3' complementary sequences (CYC sequences). This 5'-3' base pairing results in an efficient initiation of reverse transcription in vitro. Transposition of a marked Ty1 element and Ty1 cDNA synthesis in yeast rely on the ability of the CYC sequences to base pair. This 5'-3' interaction is also supported by phylogenic analysis of all full-length Ty1 and Ty2 elements present in the Saccharomyces cerevisiae genome. These novel findings lead us to propose that circularization of the Ty1 genomic RNA controls initiation of reverse transcription and may limit reverse transcription of defective retroelements.},
note = {0261-4189
Journal Article},
keywords = {*Gene, *Transcription, Acid, cerevisiae/*genetics, Complementary/biosynthesis, Conformation, DNA, Expression, Fungal, Fungal/chemistry/*metabolism, Genetic, Gov't, in, Messenger/chemistry/*metabolism, Non-U.S., Nucleic, Phylogeny, Regulation, Retroelements/*genetics, RNA, Saccharomyces, Support, vitro},
pubstate = {published},
tppubtype = {article}
}
LTR-retrotransposons are abundant components of all eukaryotic genomes and appear to be key players in their evolution. They share with retroviruses a reverse transcription step during their replication cycle. To better understand the replication of retrotransposons as well as their similarities to and differences from retroviruses, we set up an in vitro model system to examine minus-strand cDNA synthesis of the yeast Ty1 LTR-retrotransposon. Results show that the 5' and 3' ends of Ty1 genomic RNA interact through 14 nucleotide 5'-3' complementary sequences (CYC sequences). This 5'-3' base pairing results in an efficient initiation of reverse transcription in vitro. Transposition of a marked Ty1 element and Ty1 cDNA synthesis in yeast rely on the ability of the CYC sequences to base pair. This 5'-3' interaction is also supported by phylogenic analysis of all full-length Ty1 and Ty2 elements present in the Saccharomyces cerevisiae genome. These novel findings lead us to propose that circularization of the Ty1 genomic RNA controls initiation of reverse transcription and may limit reverse transcription of defective retroelements.
Perederina A., Nevskaya N., Nikonov O., Nikulin A., Dumas P., Yao M., Tanaka I., Garber M., Gongadze G., Nikonov S.
Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex Article de journal
Dans: RNA, vol. 8, non 12, p. 1548-57, 2002, (1355-8382 Journal Article).
Résumé | BibTeX | Étiquettes: 5S/*chemistry/*metabolism, Acid, Amino, Bacterial, Base, Binding, Bonding, coli/genetics, Conformation, Data, Escherichia, Fragments/chemistry/metabolism, Gov't, Hydrogen, Models, Molecular, Non-U.S., Nucleic, Peptide, Protein, Proteins/*chemistry/*metabolism, Proteins/chemistry/metabolism, Ribosomal, RNA, Sequence, Sites, Support
@article{,
title = {Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex},
author = { A. Perederina and N. Nevskaya and O. Nikonov and A. Nikulin and P. Dumas and M. Yao and I. Tanaka and M. Garber and G. Gongadze and S. Nikonov},
year = {2002},
date = {2002-01-01},
journal = {RNA},
volume = {8},
number = {12},
pages = {1548-57},
abstract = {The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit.},
note = {1355-8382
Journal Article},
keywords = {5S/*chemistry/*metabolism, Acid, Amino, Bacterial, Base, Binding, Bonding, coli/genetics, Conformation, Data, Escherichia, Fragments/chemistry/metabolism, Gov't, Hydrogen, Models, Molecular, Non-U.S., Nucleic, Peptide, Protein, Proteins/*chemistry/*metabolism, Proteins/chemistry/metabolism, Ribosomal, RNA, Sequence, Sites, Support},
pubstate = {published},
tppubtype = {article}
}
The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit.
2001
Carnicelli D., Brigotti M., Rizzi S., Keith G., Montanaro L., Sperti S.
Nucleotides U28-A42 and A37 in unmodified yeast tRNA(Trp) as negative identity elements for bovine tryptophanyl-tRNA synthetase Article de journal
Dans: FEBS Lett, vol. 492, non 3, p. 238-41, 2001, (0014-5793 Journal Article).
Résumé | BibTeX | Étiquettes: Acid, Adenine/chemistry, Animals, Base, Cattle, cerevisiae/genetics, Conformation, Data, Fungal/genetics/metabolism, Gov't, Kinetics, Ligase/*metabolism, Molecular, Non-U.S., Nucleic, RNA, Saccharomyces, Sequence, Species, Specificity, Substrate, Support, Transfer, Trp/chemistry/*metabolism, Tryptophan-tRNA, Uridine/chemistry
@article{,
title = {Nucleotides U28-A42 and A37 in unmodified yeast tRNA(Trp) as negative identity elements for bovine tryptophanyl-tRNA synthetase},
author = { D. Carnicelli and M. Brigotti and S. Rizzi and G. Keith and L. Montanaro and S. Sperti},
year = {2001},
date = {2001-01-01},
journal = {FEBS Lett},
volume = {492},
number = {3},
pages = {238-41},
abstract = {Wild-type bovine and yeast tRNA(Trp) are efficiently aminoacylated by tryptophanyl-tRNA synthetase both from beef and from yeast. Upon loss of modified bases in the synthetic transcripts, mammalian tRNA(Trp) retains the double recognition by the two synthetases, while yeast tRNA(Trp) loses its substrate properties for the bovine enzyme and is recognised only by the cognate synthetase. By testing chimeric bovine-yeast transcripts with tryptophanyl-tRNA synthetase purified from beef pancreas, the nucleotides responsible for the loss of charging of the synthetic yeast transcript have been localised in the anticodon arm. A complete loss of charging akin to that observed with the yeast transcript requires substitution in the bovine backbone of G37 in the anticodon loop with yeast A37 and of C28-G42 in the anticodon stem with yeast U28-A42. Since A37 does not prevent aminoacylation of the wild-type yeast tRNA(Trp) by the beef enzyme, a negative combination apparently emerges in the synthetic transcript after unmasking of U28 by loss of pseudourydilation.},
note = {0014-5793
Journal Article},
keywords = {Acid, Adenine/chemistry, Animals, Base, Cattle, cerevisiae/genetics, Conformation, Data, Fungal/genetics/metabolism, Gov't, Kinetics, Ligase/*metabolism, Molecular, Non-U.S., Nucleic, RNA, Saccharomyces, Sequence, Species, Specificity, Substrate, Support, Transfer, Trp/chemistry/*metabolism, Tryptophan-tRNA, Uridine/chemistry},
pubstate = {published},
tppubtype = {article}
}
Wild-type bovine and yeast tRNA(Trp) are efficiently aminoacylated by tryptophanyl-tRNA synthetase both from beef and from yeast. Upon loss of modified bases in the synthetic transcripts, mammalian tRNA(Trp) retains the double recognition by the two synthetases, while yeast tRNA(Trp) loses its substrate properties for the bovine enzyme and is recognised only by the cognate synthetase. By testing chimeric bovine-yeast transcripts with tryptophanyl-tRNA synthetase purified from beef pancreas, the nucleotides responsible for the loss of charging of the synthetic yeast transcript have been localised in the anticodon arm. A complete loss of charging akin to that observed with the yeast transcript requires substitution in the bovine backbone of G37 in the anticodon loop with yeast A37 and of C28-G42 in the anticodon stem with yeast U28-A42. Since A37 does not prevent aminoacylation of the wild-type yeast tRNA(Trp) by the beef enzyme, a negative combination apparently emerges in the synthetic transcript after unmasking of U28 by loss of pseudourydilation.
Moine H., Mandel J. L.
Biomedicine. Do G quartets orchestrate fragile X pathology? Article de journal
Dans: Science, vol. 294, non 5551, p. 2487-8, 2001, (0036-8075 Journal Article).
BibTeX | Étiquettes: Acid, Analysis, Animals, Array, Binding, Brain/metabolism, Conformation, Crystallography, Expression, Fragile, Gene, Genetic, Human, Messenger/*chemistry/genetics/*metabolism, Mice, Nerve, Nucleic, Oligonucleotide, Protein, Proteins/chemistry/genetics/*metabolism, Regions, Regulation, RNA, Sequence, Sites, structure, Synapses/physiology, Syndrome/genetics/*metabolism, Tertiary, Tissue, Translation, Untranslated, X, X-Ray
@article{,
title = {Biomedicine. Do G quartets orchestrate fragile X pathology?},
author = { H. Moine and J. L. Mandel},
year = {2001},
date = {2001-01-01},
journal = {Science},
volume = {294},
number = {5551},
pages = {2487-8},
note = {0036-8075
Journal Article},
keywords = {Acid, Analysis, Animals, Array, Binding, Brain/metabolism, Conformation, Crystallography, Expression, Fragile, Gene, Genetic, Human, Messenger/*chemistry/genetics/*metabolism, Mice, Nerve, Nucleic, Oligonucleotide, Protein, Proteins/chemistry/genetics/*metabolism, Regions, Regulation, RNA, Sequence, Sites, structure, Synapses/physiology, Syndrome/genetics/*metabolism, Tertiary, Tissue, Translation, Untranslated, X, X-Ray},
pubstate = {published},
tppubtype = {article}
}
Wilhelm M., Wilhelm F. X.
Reverse transcription of retroviruses and LTR retrotransposons Article de journal
Dans: Cell Mol Life Sci, vol. 58, non 9, p. 1246-62, 2001, (1420-682x Journal Article Review Review, Academic).
Résumé | BibTeX | Étiquettes: *Retroelements, *Terminal, Acid, Alignment, Amino, Animals, Base, Conformation, Data, DNA, Homology, Human, Molecular, Nucleic, Polymerase/*chemistry/*metabolism, Repeat, Retroviridae/*enzymology/*genetics, RNA-Directed, Sequence, Sequences
@article{,
title = {Reverse transcription of retroviruses and LTR retrotransposons},
author = { M. Wilhelm and F. X. Wilhelm},
year = {2001},
date = {2001-01-01},
journal = {Cell Mol Life Sci},
volume = {58},
number = {9},
pages = {1246-62},
abstract = {Retroelements are mobile genetic entities that replicate via reverse transcription of a template RNA. A key component to the life cycle of these elements is the enzyme reverse transcriptase (RT), which copies the single-stranded genomic RNA of the element into a linear double-stranded DNA that is ultimately integrated into the host genome by the element-encoded integrase. RT is a multifunctionnal enzyme which possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity that specifically degrades the RNA strand of RNA-DNA duplexes. At some stages of the replication a strand-displacement activity of RT is also necessary. All activities are essential for the conversion of single-stranded genomic RNA into the double-stranded preintegrative DNA. This review focuses on the role of RT in the different steps of the replication process of retroelements. The features of retrotransposon replication which differ from the retroviral ones will be emphasized. In a second part of the review, the biochemical and enzymatic properties of two newly characterized retrotransposon RTs will be described. The role of the integrase domain in reverse transcriptase activity of some retroviral and retrotransposon RTs will be discussed.},
note = {1420-682x
Journal Article
Review
Review, Academic},
keywords = {*Retroelements, *Terminal, Acid, Alignment, Amino, Animals, Base, Conformation, Data, DNA, Homology, Human, Molecular, Nucleic, Polymerase/*chemistry/*metabolism, Repeat, Retroviridae/*enzymology/*genetics, RNA-Directed, Sequence, Sequences},
pubstate = {published},
tppubtype = {article}
}
Retroelements are mobile genetic entities that replicate via reverse transcription of a template RNA. A key component to the life cycle of these elements is the enzyme reverse transcriptase (RT), which copies the single-stranded genomic RNA of the element into a linear double-stranded DNA that is ultimately integrated into the host genome by the element-encoded integrase. RT is a multifunctionnal enzyme which possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity that specifically degrades the RNA strand of RNA-DNA duplexes. At some stages of the replication a strand-displacement activity of RT is also necessary. All activities are essential for the conversion of single-stranded genomic RNA into the double-stranded preintegrative DNA. This review focuses on the role of RT in the different steps of the replication process of retroelements. The features of retrotransposon replication which differ from the retroviral ones will be emphasized. In a second part of the review, the biochemical and enzymatic properties of two newly characterized retrotransposon RTs will be described. The role of the integrase domain in reverse transcriptase activity of some retroviral and retrotransposon RTs will be discussed.
2000
Wilhelm M., Boutabout M., Wilhelm F. X.
Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities Article de journal
Dans: Biochem J, vol. 348, non Pt 2, p. 337-42, 2000, (0264-6021 Journal Article).
Résumé | BibTeX | Étiquettes: &, Acid, affinity, Alignment, Amino, Calf, cerevisiae/*enzymology/*genetics, Chromatography, Cloning, Codon, coli, Comparative, Data, DNA, DNA/metabolism, Escherichia, Frames, Fusion, Genetic, Gov't, H, Heteroduplexes/metabolism, HIV-1, Homology, Integrases/chemistry/metabolism, Kinetics, Molecular, Non-U.S., Nucleic, Open, Polymerase/chemistry/isolation, Proteins/chemistry/isolation, purification/*metabolism, purification/metabolism, Reading, Recombinant, Retroelements/*genetics, Reverse, Ribonuclease, RNA-Directed, RNA/metabolism, Saccharomyces, Sequence, Study, Support, Templates, Terminator, Thymus/isolation, Transcriptase/chemistry
@article{,
title = {Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities},
author = { M. Wilhelm and M. Boutabout and F. X. Wilhelm},
year = {2000},
date = {2000-01-01},
journal = {Biochem J},
volume = {348},
number = {Pt 2},
pages = {337-42},
abstract = {Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl(2), NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.},
note = {0264-6021
Journal Article},
keywords = {&, Acid, affinity, Alignment, Amino, Calf, cerevisiae/*enzymology/*genetics, Chromatography, Cloning, Codon, coli, Comparative, Data, DNA, DNA/metabolism, Escherichia, Frames, Fusion, Genetic, Gov't, H, Heteroduplexes/metabolism, HIV-1, Homology, Integrases/chemistry/metabolism, Kinetics, Molecular, Non-U.S., Nucleic, Open, Polymerase/chemistry/isolation, Proteins/chemistry/isolation, purification/*metabolism, purification/metabolism, Reading, Recombinant, Retroelements/*genetics, Reverse, Ribonuclease, RNA-Directed, RNA/metabolism, Saccharomyces, Sequence, Study, Support, Templates, Terminator, Thymus/isolation, Transcriptase/chemistry},
pubstate = {published},
tppubtype = {article}
}
Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl(2), NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.
1999
Auxilien S., Keith G., Grice S. F. Le, Darlix J. L.
Role of post-transcriptional modifications of primer tRNALys,3 in the fidelity and efficacy of plus strand DNA transfer during HIV-1 reverse transcription Article de journal
Dans: J Biol Chem, vol. 274, non 7, p. 4412-20, 1999, (0021-9258 Journal Article).
Résumé | BibTeX | Étiquettes: *RNA, *Transcription, Acid, Base, Calf, Conformation, Data, DNA, Genetic, Gov't, H, HIV-1, HIV-1/*physiology, Lys/*metabolism, Molecular, Non-U.S., Nucleic, post-transcriptional, Processing, Reverse, Ribonuclease, RNA, Sequence, Support, Templates, Thymus/metabolism, Transcriptase/metabolism, Transfer, Viral/*metabolism, Viral/metabolism
@article{,
title = {Role of post-transcriptional modifications of primer tRNALys,3 in the fidelity and efficacy of plus strand DNA transfer during HIV-1 reverse transcription},
author = { S. Auxilien and G. Keith and S. F. Le Grice and J. L. Darlix},
year = {1999},
date = {1999-01-01},
journal = {J Biol Chem},
volume = {274},
number = {7},
pages = {4412-20},
abstract = {During HIV reverse transcription, (+) strand DNA synthesis is primed by an RNase H-resistant sequence, the polypurine tract, and continues as far as a 18-nt double-stranded RNA region corresponding to the 3' end of tRNALys,3 hybridized to the viral primer binding site (PBS). Before (+) strand DNA transfer, reverse transcriptase (RT) needs to unwind the double-stranded tRNA-PBS RNA in order to reverse-transcribe the 3' end of primer tRNALys,3. Since the detailed mechanism of (+) strand DNA transfer remains incompletely understood, we developed an in vitro system to closely examine this mechanism, composed of HIV 5' RNA, natural modified tRNALys,3, synthetic unmodified tRNALys,3 or oligonucleotides (RNA or DNA) complementary to the PBS, as well as the viral proteins RT and nucleocapsid protein (NCp7). Prior to (+) strand DNA transfer, RT stalls at the double-stranded tRNA-PBS RNA complex and is able to reverse-transcribe modified nucleosides of natural tRNALys,3. Modified nucleoside m1A-58 of natural tRNALys,3 is only partially effective as a stop signal, as RT can transcribe as far as the hyper-modified adenosine (ms2t6A-37) in the anticodon loop. m1A-58 is almost always transcribed into A, whereas other modified nucleosides are transcribed correctly, except for m7G-46, which is sometimes transcribed into T. In contrast, synthetic tRNALys,3, an RNA PBS primer, and a DNA PBS primer are completely reverse-transcribed. In the presence of an acceptor template, (+) strand DNA transfer is efficient only with templates containing natural tRNALys,3 or the RNA PBS primer. Sequence analysis of transfer products revealed frequent errors at the transfer site with synthetic tRNALys,3, not observed with natural tRNALys,3. Thus, modified nucleoside m1A-58, present in all retroviral tRNA primers, appears to be important for both efficacy and fidelity of (+) strand DNA transfer. We show that other factors such as the nature of the (-) PBS of the acceptor template and the RNase H activity of RT also influence the efficacy of (+) strand DNA transfer.},
note = {0021-9258
Journal Article},
keywords = {*RNA, *Transcription, Acid, Base, Calf, Conformation, Data, DNA, Genetic, Gov't, H, HIV-1, HIV-1/*physiology, Lys/*metabolism, Molecular, Non-U.S., Nucleic, post-transcriptional, Processing, Reverse, Ribonuclease, RNA, Sequence, Support, Templates, Thymus/metabolism, Transcriptase/metabolism, Transfer, Viral/*metabolism, Viral/metabolism},
pubstate = {published},
tppubtype = {article}
}
During HIV reverse transcription, (+) strand DNA synthesis is primed by an RNase H-resistant sequence, the polypurine tract, and continues as far as a 18-nt double-stranded RNA region corresponding to the 3' end of tRNALys,3 hybridized to the viral primer binding site (PBS). Before (+) strand DNA transfer, reverse transcriptase (RT) needs to unwind the double-stranded tRNA-PBS RNA in order to reverse-transcribe the 3' end of primer tRNALys,3. Since the detailed mechanism of (+) strand DNA transfer remains incompletely understood, we developed an in vitro system to closely examine this mechanism, composed of HIV 5' RNA, natural modified tRNALys,3, synthetic unmodified tRNALys,3 or oligonucleotides (RNA or DNA) complementary to the PBS, as well as the viral proteins RT and nucleocapsid protein (NCp7). Prior to (+) strand DNA transfer, RT stalls at the double-stranded tRNA-PBS RNA complex and is able to reverse-transcribe modified nucleosides of natural tRNALys,3. Modified nucleoside m1A-58 of natural tRNALys,3 is only partially effective as a stop signal, as RT can transcribe as far as the hyper-modified adenosine (ms2t6A-37) in the anticodon loop. m1A-58 is almost always transcribed into A, whereas other modified nucleosides are transcribed correctly, except for m7G-46, which is sometimes transcribed into T. In contrast, synthetic tRNALys,3, an RNA PBS primer, and a DNA PBS primer are completely reverse-transcribed. In the presence of an acceptor template, (+) strand DNA transfer is efficient only with templates containing natural tRNALys,3 or the RNA PBS primer. Sequence analysis of transfer products revealed frequent errors at the transfer site with synthetic tRNALys,3, not observed with natural tRNALys,3. Thus, modified nucleoside m1A-58, present in all retroviral tRNA primers, appears to be important for both efficacy and fidelity of (+) strand DNA transfer. We show that other factors such as the nature of the (-) PBS of the acceptor template and the RNase H activity of RT also influence the efficacy of (+) strand DNA transfer.
Wilhelm M., Boutabout M., Heyman T., Wilhelm F. X.
Reverse transcription of the yeast Ty1 retrotransposon: the mode of first strand transfer is either intermolecular or intramolecular Article de journal
Dans: J Mol Biol, vol. 288, non 4, p. 505-10, 1999, (0022-2836 Journal Article).
Résumé | BibTeX | Étiquettes: *Retroelements, *Transcription, Acid, Base, cerevisiae/*genetics, DNA, Genetic, Gov't, Non-U.S., Nucleic, Repetitive, Saccharomyces, Sequence, Sequences, Single-Stranded/genetics, Support
@article{,
title = {Reverse transcription of the yeast Ty1 retrotransposon: the mode of first strand transfer is either intermolecular or intramolecular},
author = { M. Wilhelm and M. Boutabout and T. Heyman and F. X. Wilhelm},
year = {1999},
date = {1999-01-01},
journal = {J Mol Biol},
volume = {288},
number = {4},
pages = {505-10},
abstract = {Replication of the yeast Ty1 retrotransposon occurs by a mechanism similar to that of retroviruses. According to the current model of retroviral reverse transcription, two strand transfers (the so-called minus-strand and plus-strand strong-stop DNA transfers) are required to produce full-length preintegrative DNA. Because two genomic RNA molecules are packaged inside the viral particles, the strand transfers can be either intra- or intermolecular. To study the mode of transfer of minus-strand strong-stop DNA during reverse transcription of the yeast Ty1 retrotransposon, we have analyzed the cDNA products that accumulate in the cytoplasmic virus-like particles of yeast cells harboring two marked Ty1 elements. Our results indicate that Ty1 minus-strand transfer occurs in a random manner with approximately similar frequencies of intra- and intermolecular transfer. It has been observed recently that intra- and intermolecular minus-strand transfer occur at similar frequencies during replication of a complex retrovirus such as HIV-1. These results together with the observation that genetic recombination occurs with a high frequency during minus-strand synthesis suggest that both packaged RNA molecules are needed for the synthesis of one minus-strand DNA.},
note = {0022-2836
Journal Article},
keywords = {*Retroelements, *Transcription, Acid, Base, cerevisiae/*genetics, DNA, Genetic, Gov't, Non-U.S., Nucleic, Repetitive, Saccharomyces, Sequence, Sequences, Single-Stranded/genetics, Support},
pubstate = {published},
tppubtype = {article}
}
Replication of the yeast Ty1 retrotransposon occurs by a mechanism similar to that of retroviruses. According to the current model of retroviral reverse transcription, two strand transfers (the so-called minus-strand and plus-strand strong-stop DNA transfers) are required to produce full-length preintegrative DNA. Because two genomic RNA molecules are packaged inside the viral particles, the strand transfers can be either intra- or intermolecular. To study the mode of transfer of minus-strand strong-stop DNA during reverse transcription of the yeast Ty1 retrotransposon, we have analyzed the cDNA products that accumulate in the cytoplasmic virus-like particles of yeast cells harboring two marked Ty1 elements. Our results indicate that Ty1 minus-strand transfer occurs in a random manner with approximately similar frequencies of intra- and intermolecular transfer. It has been observed recently that intra- and intermolecular minus-strand transfer occur at similar frequencies during replication of a complex retrovirus such as HIV-1. These results together with the observation that genetic recombination occurs with a high frequency during minus-strand synthesis suggest that both packaged RNA molecules are needed for the synthesis of one minus-strand DNA.
1998
Brigotti M., Keith G., Pallanca A., Carnicelli D., Alvergna P., Dirheimer G., Montanaro L., Sperti S.
Identification of the tRNAs which up-regulate agrostin, barley RIP and PAP-S, three ribosome-inactivating proteins of plant origin Article de journal
Dans: FEBS Lett, vol. 431, non 2, p. 259-62, 1998, (0014-5793 Journal Article).
Résumé | BibTeX | Étiquettes: &, Acid, Adenosine, Base, Conformation, Data, effects/*metabolism, Gov't, Hordeum/metabolism, Hydrolases/*metabolism, Molecular, N-Glycosyl, Non-U.S., Nucleic, Plant, Plant/chemistry/isolation, Proteins/drug, purification/*metabolism, RNA, Sequence, Support, Transfer/chemistry/isolation, Triphosphate/pharmacology, Up-Regulation
@article{,
title = {Identification of the tRNAs which up-regulate agrostin, barley RIP and PAP-S, three ribosome-inactivating proteins of plant origin},
author = { M. Brigotti and G. Keith and A. Pallanca and D. Carnicelli and P. Alvergna and G. Dirheimer and L. Montanaro and S. Sperti},
year = {1998},
date = {1998-01-01},
journal = {FEBS Lett},
volume = {431},
number = {2},
pages = {259-62},
abstract = {Ribosome-inactivating proteins (RIP) are RNA-N-glycosidases widely diffused in plants which depurinate ribosomal RNA at a specific universally conserved position, A4324 in rat ribosomes. A small group of RIPs (cofactor-dependent RIPs) require ATP and tRNA to reach maximal activity on isolated ribosomes. The tRNA which stimulates gelonin was identified as tRNA(Trp). The present paper reports the identification of three other tRNAs which stimulate agrostin (tRNA(Ala)), barley RIP (tRNA(Ala), tRNA(Val)) and PAP-S (tRNA(Gly)), while for tritin-S no particular stimulating tRNA emerged. The sequences of tRNA(Val) and tRNA(Gly) correspond to the already known ones (rabbit and man, respectively). The tRNA(Ala) (anticodon IGC) identifies a new isoacceptor. Only the stimulating activity of the tRNA(Ala) for agrostin approaches the specificity previously observed for the couple gelonin-tRNA(Trp).},
note = {0014-5793
Journal Article},
keywords = {&, Acid, Adenosine, Base, Conformation, Data, effects/*metabolism, Gov't, Hordeum/metabolism, Hydrolases/*metabolism, Molecular, N-Glycosyl, Non-U.S., Nucleic, Plant, Plant/chemistry/isolation, Proteins/drug, purification/*metabolism, RNA, Sequence, Support, Transfer/chemistry/isolation, Triphosphate/pharmacology, Up-Regulation},
pubstate = {published},
tppubtype = {article}
}
Ribosome-inactivating proteins (RIP) are RNA-N-glycosidases widely diffused in plants which depurinate ribosomal RNA at a specific universally conserved position, A4324 in rat ribosomes. A small group of RIPs (cofactor-dependent RIPs) require ATP and tRNA to reach maximal activity on isolated ribosomes. The tRNA which stimulates gelonin was identified as tRNA(Trp). The present paper reports the identification of three other tRNAs which stimulate agrostin (tRNA(Ala)), barley RIP (tRNA(Ala), tRNA(Val)) and PAP-S (tRNA(Gly)), while for tritin-S no particular stimulating tRNA emerged. The sequences of tRNA(Val) and tRNA(Gly) correspond to the already known ones (rabbit and man, respectively). The tRNA(Ala) (anticodon IGC) identifies a new isoacceptor. Only the stimulating activity of the tRNA(Ala) for agrostin approaches the specificity previously observed for the couple gelonin-tRNA(Trp).
Friant S., Heyman T., Bystrom A. S., Wilhelm M., Wilhelm F. X.
Interactions between Ty1 retrotransposon RNA and the T and D regions of the tRNA(iMet) primer are required for initiation of reverse transcription in vivo Article de journal
Dans: Mol Cell Biol, vol. 18, non 2, p. 799-806, 1998, (0270-7306 Journal Article).
Résumé | BibTeX | Étiquettes: *Retroelements, *Transcription, Acid, Base, Binding, cerevisiae, Conformation, Data, DNA, Fungal/*metabolism, Fungal/biosynthesis, Genetic, Gov't, Met/*metabolism, Molecular, Mutagenesis, Non-U.S., Nucleic, Primers, Replication, RNA, Saccharomyces, Sequence, Sites, Support, Transfer
@article{,
title = {Interactions between Ty1 retrotransposon RNA and the T and D regions of the tRNA(iMet) primer are required for initiation of reverse transcription in vivo},
author = { S. Friant and T. Heyman and A. S. Bystrom and M. Wilhelm and F. X. Wilhelm},
year = {1998},
date = {1998-01-01},
journal = {Mol Cell Biol},
volume = {18},
number = {2},
pages = {799-806},
abstract = {Reverse transcription of the Saccharomyces cerevisiae Ty1 retrotransposon is primed by tRNA(iMet) base paired to the primer binding site (PBS) near the 5' end of Ty1 genomic RNA. The 10-nucleotide PBS is complementary to the last 10 nucleotides of the acceptor stem of tRNA(iMet). A structural probing study of the interactions between the Ty1 RNA template and the tRNA(iMet) primer showed that besides interactions between the PBS and the 3' end of tRNA(iMet), three short regions of Ty1 RNA, named boxes 0, 1, and 2.1, interact with the T and D stems and loops of tRNA(iMet). To determine if these sequences are important for the reverse transcription pathway of the Ty1 retrotransposon, mutant Ty1 elements and tRNA(iMet) were tested for the ability to support transposition. We show that the Ty1 boxes and the complementary sequences in the T and D stems and loops of tRNA(iMet) contain bases that are critical for Ty1 retrotransposition. Disruption of 1 or 2 bp between tRNA(iMet) and box 0, 1, or 2.1 dramatically decreases the level of transposition. Compensatory mutations which restore base pairing between the primer and the template restore transposition. Analysis of the reverse transcription intermediates generated inside Ty1 virus-like particles indicates that initiation of minus-strand strong-stop DNA synthesis is affected by mutations disrupting complementarity between Ty1 RNA and primer tRNA(iMet).},
note = {0270-7306
Journal Article},
keywords = {*Retroelements, *Transcription, Acid, Base, Binding, cerevisiae, Conformation, Data, DNA, Fungal/*metabolism, Fungal/biosynthesis, Genetic, Gov't, Met/*metabolism, Molecular, Mutagenesis, Non-U.S., Nucleic, Primers, Replication, RNA, Saccharomyces, Sequence, Sites, Support, Transfer},
pubstate = {published},
tppubtype = {article}
}
Reverse transcription of the Saccharomyces cerevisiae Ty1 retrotransposon is primed by tRNA(iMet) base paired to the primer binding site (PBS) near the 5' end of Ty1 genomic RNA. The 10-nucleotide PBS is complementary to the last 10 nucleotides of the acceptor stem of tRNA(iMet). A structural probing study of the interactions between the Ty1 RNA template and the tRNA(iMet) primer showed that besides interactions between the PBS and the 3' end of tRNA(iMet), three short regions of Ty1 RNA, named boxes 0, 1, and 2.1, interact with the T and D stems and loops of tRNA(iMet). To determine if these sequences are important for the reverse transcription pathway of the Ty1 retrotransposon, mutant Ty1 elements and tRNA(iMet) were tested for the ability to support transposition. We show that the Ty1 boxes and the complementary sequences in the T and D stems and loops of tRNA(iMet) contain bases that are critical for Ty1 retrotransposition. Disruption of 1 or 2 bp between tRNA(iMet) and box 0, 1, or 2.1 dramatically decreases the level of transposition. Compensatory mutations which restore base pairing between the primer and the template restore transposition. Analysis of the reverse transcription intermediates generated inside Ty1 virus-like particles indicates that initiation of minus-strand strong-stop DNA synthesis is affected by mutations disrupting complementarity between Ty1 RNA and primer tRNA(iMet).
Gabus C., Ficheux D., Rau M., Keith G., Sandmeyer S., Darlix J. L.
The yeast Ty3 retrotransposon contains a 5'-3' bipartite primer-binding site and encodes nucleocapsid protein NCp9 functionally homologous to HIV-1 NCp7 Article de journal
Dans: EMBO J, vol. 17, non 16, p. 4873-80, 1998, (0261-4189 Journal Article).
Résumé | BibTeX | Étiquettes: *Capsid, *Retroelements, Acid, Base, Binding, Capsid/*genetics, cerevisiae/*genetics, dimerization, gag/*genetics, Gene, Gov't, Homology, Met/genetics/*metabolism, Non-U.S., Nucleic, P.H.S., Products, Proteins, RNA, Saccharomyces, Sequence, Sites, Support, Transfer, U.S.
@article{,
title = {The yeast Ty3 retrotransposon contains a 5'-3' bipartite primer-binding site and encodes nucleocapsid protein NCp9 functionally homologous to HIV-1 NCp7},
author = { C. Gabus and D. Ficheux and M. Rau and G. Keith and S. Sandmeyer and J. L. Darlix},
year = {1998},
date = {1998-01-01},
journal = {EMBO J},
volume = {17},
number = {16},
pages = {4873-80},
abstract = {Retroviruses, including HIV-1 and the distantly related yeast retroelement Ty3, all encode a nucleoprotein required for virion structure and replication. During an in vitro comparison of HIV-1 and Ty3 nucleoprotein function in RNA dimerization and cDNA synthesis, we discovered a bipartite primer-binding site (PBS) for Ty3 composed of sequences located at opposite ends of the genome. Ty3 cDNA synthesis requires the 3' PBS for primer tRNAiMet annealing to the genomic RNA, and the 5' PBS, in cis or in trans, as the reverse transcription start site. Ty3 RNA alone is unable to dimerize, but formation of dimeric tRNAiMet bound to the PBS was found to direct dimerization of Ty3 RNA-tRNAiMet. Interestingly, HIV-1 nucleocapsid protein NCp7 and Ty3 NCp9 were interchangeable using HIV-1 and Ty3 RNA template-primer systems. Our findings impact on the understanding of non-canonical reverse transcription as well as on the use of Ty3 systems to screen for anti-NCp7 drugs.},
note = {0261-4189
Journal Article},
keywords = {*Capsid, *Retroelements, Acid, Base, Binding, Capsid/*genetics, cerevisiae/*genetics, dimerization, gag/*genetics, Gene, Gov't, Homology, Met/genetics/*metabolism, Non-U.S., Nucleic, P.H.S., Products, Proteins, RNA, Saccharomyces, Sequence, Sites, Support, Transfer, U.S.},
pubstate = {published},
tppubtype = {article}
}
Retroviruses, including HIV-1 and the distantly related yeast retroelement Ty3, all encode a nucleoprotein required for virion structure and replication. During an in vitro comparison of HIV-1 and Ty3 nucleoprotein function in RNA dimerization and cDNA synthesis, we discovered a bipartite primer-binding site (PBS) for Ty3 composed of sequences located at opposite ends of the genome. Ty3 cDNA synthesis requires the 3' PBS for primer tRNAiMet annealing to the genomic RNA, and the 5' PBS, in cis or in trans, as the reverse transcription start site. Ty3 RNA alone is unable to dimerize, but formation of dimeric tRNAiMet bound to the PBS was found to direct dimerization of Ty3 RNA-tRNAiMet. Interestingly, HIV-1 nucleocapsid protein NCp7 and Ty3 NCp9 were interchangeable using HIV-1 and Ty3 RNA template-primer systems. Our findings impact on the understanding of non-canonical reverse transcription as well as on the use of Ty3 systems to screen for anti-NCp7 drugs.
1997
Wilhelm M., Heyman T., Friant S., Wilhelm F. X.
Heterogeneous terminal structure of Ty1 and Ty3 reverse transcripts Article de journal
Dans: Nucleic Acids Res, vol. 25, non 11, p. 2161-6, 1997, (0305-1048 Journal Article).
Résumé | BibTeX | Étiquettes: *Nucleic, *Transcription, Acid, Calf, Chain, Conformation, DNA, Fungal/*chemistry/metabolism, Genetic, Gov't, H, Hybridization, Non-U.S., Nucleic, Plasmids/chemistry/genetics/metabolism, Polymerase, Reaction, Replication, Retroelements/*genetics, Ribonuclease, RNA, Support, Thymus/metabolism, Transfer/chemistry
@article{,
title = {Heterogeneous terminal structure of Ty1 and Ty3 reverse transcripts},
author = { M. Wilhelm and T. Heyman and S. Friant and F. X. Wilhelm},
year = {1997},
date = {1997-01-01},
journal = {Nucleic Acids Res},
volume = {25},
number = {11},
pages = {2161-6},
abstract = {A specific terminal structure of preintegrative DNA is required for transposition of retroviruses and LTR-retrotransposons. We have used an anchored PCR technique to map the 3'ends of DNA intermediates synthesized inside yeast Ty1 and Ty3 retrotransposon virus-like particles. We find that, unlike retroviruses, Ty1 replicated DNA does not have two extra base pairs at its 3'ends. In contrast some Ty3 preintegrative DNA molecules have two extra nucleotides at the 3'end of upstream and downstream long terminal repeats. Moreover we find that some molecules of replicated Ty3 DNA have more than two extra nucleotides at the 3'end of the upstream LTR. This observation could be accounted for by imprecise RNAse H cutting of the PPT sequence. The site of Ty1 and Ty3 plus-strand strong-stop DNA termination was also examined. Our results confirm that the prominent Ty1 and Ty3 plus-strand strong-stop molecules harbor 12 tRNA templated bases but also show that some Ty1 and Ty3 plus-strand strong-stop DNA molecules harbor less tRNA templated bases. We propose that these less than full length plus-strand molecules could be active intermediates in Ty retrotransposon replication.},
note = {0305-1048
Journal Article},
keywords = {*Nucleic, *Transcription, Acid, Calf, Chain, Conformation, DNA, Fungal/*chemistry/metabolism, Genetic, Gov't, H, Hybridization, Non-U.S., Nucleic, Plasmids/chemistry/genetics/metabolism, Polymerase, Reaction, Replication, Retroelements/*genetics, Ribonuclease, RNA, Support, Thymus/metabolism, Transfer/chemistry},
pubstate = {published},
tppubtype = {article}
}
A specific terminal structure of preintegrative DNA is required for transposition of retroviruses and LTR-retrotransposons. We have used an anchored PCR technique to map the 3'ends of DNA intermediates synthesized inside yeast Ty1 and Ty3 retrotransposon virus-like particles. We find that, unlike retroviruses, Ty1 replicated DNA does not have two extra base pairs at its 3'ends. In contrast some Ty3 preintegrative DNA molecules have two extra nucleotides at the 3'end of upstream and downstream long terminal repeats. Moreover we find that some molecules of replicated Ty3 DNA have more than two extra nucleotides at the 3'end of the upstream LTR. This observation could be accounted for by imprecise RNAse H cutting of the PPT sequence. The site of Ty1 and Ty3 plus-strand strong-stop DNA termination was also examined. Our results confirm that the prominent Ty1 and Ty3 plus-strand strong-stop molecules harbor 12 tRNA templated bases but also show that some Ty1 and Ty3 plus-strand strong-stop DNA molecules harbor less tRNA templated bases. We propose that these less than full length plus-strand molecules could be active intermediates in Ty retrotransposon replication.
1996
Friant S., Heyman T., Wilhelm F. X., Wilhelm M.
Role of RNA primers in initiation of minus-strand and plus-strand DNA synthesis of the yeast retrotransposon Ty1 Article de journal
Dans: Biochimie, vol. 78, non 7, p. 674-80, 1996, (0300-9084 Journal Article).
Résumé | BibTeX | Étiquettes: *DNA, Acid, Bacterial/*metabolism, Complementary/metabolism, Conformation, Data, DNA, Elements, Gov't, Molecular, Mutagenesis, Non-U.S., Nucleic, Replication, RNA, RNA/*metabolism, Sequence, Support, Transposable
@article{,
title = {Role of RNA primers in initiation of minus-strand and plus-strand DNA synthesis of the yeast retrotransposon Ty1},
author = { S. Friant and T. Heyman and F. X. Wilhelm and M. Wilhelm},
year = {1996},
date = {1996-01-01},
journal = {Biochimie},
volume = {78},
number = {7},
pages = {674-80},
abstract = {The Ty1 retrotransposon of the yeast Saccharomyces cerevisiae is a long terminal repeat mobile genetic element that transposes through an RNA intermediate. Initiation of minus-strand and plus-strand DNA synthesis are two critical steps during reverse transcription of the retrotransposon genome. Initiation of minus-strand DNA synthesis of the Ty1 element is primed by the cytoplasmic initiator methionine tRNA base paired to the primer binding site near the 5' end of the genomic RNA. A structural probing study of the primer tRNA-Ty1 RNA binary complex reveals that besides interactions between the primer binding site and the last 10 nucleotides at the 3' end of the primer tRNA, three short regions of Ty1 RNA named box 0, box 1 and box 2.1 interact with the T and D stems and loops of the primer tRNA. Some in vivo results underline the functional importance of the nucleotide sequence of the boxes and suggest that extended interactions between genomic Ty1 RNA and the primer tRNA play a role in the reverse transcription pathway. Plus-strand DNA synthesis is initiated from an RNase H resistant oligoribonucleotide spanning a purine-rich sequence, the polypurine tract (PPT). Two sites of initiation located at the 5' boundary of the 3' long terminal repeat (PPT1) and near the middle of the TyB (pol) gene in the integrase coding sequence (PPT2) have been identified in the genome of Ty1. The two PPTs have an identical sequence, TGGGTGGTA. Mutations replacing purines by pyrimidines in this sequence significantly diminish or abolish initiation of plus-strand DNA synthesis. Ty1 elements bearing a mutated PPT2 sequence are not defective for transposition whereas mutations in PPT1 abolish transposition.},
note = {0300-9084
Journal Article},
keywords = {*DNA, Acid, Bacterial/*metabolism, Complementary/metabolism, Conformation, Data, DNA, Elements, Gov't, Molecular, Mutagenesis, Non-U.S., Nucleic, Replication, RNA, RNA/*metabolism, Sequence, Support, Transposable},
pubstate = {published},
tppubtype = {article}
}
The Ty1 retrotransposon of the yeast Saccharomyces cerevisiae is a long terminal repeat mobile genetic element that transposes through an RNA intermediate. Initiation of minus-strand and plus-strand DNA synthesis are two critical steps during reverse transcription of the retrotransposon genome. Initiation of minus-strand DNA synthesis of the Ty1 element is primed by the cytoplasmic initiator methionine tRNA base paired to the primer binding site near the 5' end of the genomic RNA. A structural probing study of the primer tRNA-Ty1 RNA binary complex reveals that besides interactions between the primer binding site and the last 10 nucleotides at the 3' end of the primer tRNA, three short regions of Ty1 RNA named box 0, box 1 and box 2.1 interact with the T and D stems and loops of the primer tRNA. Some in vivo results underline the functional importance of the nucleotide sequence of the boxes and suggest that extended interactions between genomic Ty1 RNA and the primer tRNA play a role in the reverse transcription pathway. Plus-strand DNA synthesis is initiated from an RNase H resistant oligoribonucleotide spanning a purine-rich sequence, the polypurine tract (PPT). Two sites of initiation located at the 5' boundary of the 3' long terminal repeat (PPT1) and near the middle of the TyB (pol) gene in the integrase coding sequence (PPT2) have been identified in the genome of Ty1. The two PPTs have an identical sequence, TGGGTGGTA. Mutations replacing purines by pyrimidines in this sequence significantly diminish or abolish initiation of plus-strand DNA synthesis. Ty1 elements bearing a mutated PPT2 sequence are not defective for transposition whereas mutations in PPT1 abolish transposition.
Friant S., Heyman T., Wilhelm M. L., Wilhelm F. X.
Extended interactions between the primer tRNAi(Met) and genomic RNA of the yeast Ty1 retrotransposon Article de journal
Dans: Nucleic Acids Res, vol. 24, non 3, p. 441-9, 1996, (0305-1048 Journal Article).
Résumé | BibTeX | Étiquettes: Acid, Base, cerevisiae, Conformation, Data, Gov't, Met/genetics/*metabolism, Molecular, Mutation, Non-U.S., Nucleic, Retroelements/*genetics, RNA, RNA/genetics/*metabolism, Saccharomyces, Sequence, structure, Support, Transfer
@article{,
title = {Extended interactions between the primer tRNAi(Met) and genomic RNA of the yeast Ty1 retrotransposon},
author = { S. Friant and T. Heyman and M. L. Wilhelm and F. X. Wilhelm},
year = {1996},
date = {1996-01-01},
journal = {Nucleic Acids Res},
volume = {24},
number = {3},
pages = {441-9},
abstract = {Reverse transcription of the yeast Ty1 retrotransposon is primed by tRNAi(Met) base paired to the primer binding site near the 5'-end of Ty1 genomic RNA. To understand the molecular basis of the tRNAi(Met)-Ty1 RNA interaction the secondary structure of the binary complex was analysed. Enzymatic probes were used to test the conformation of tRNAi(Met) and of Ty1 RNA in the free form and in the complex. A secondary structure model of the tRNAi(Met) Ty1 RNA complex consistent with the probing data was constructed with the help of a computer program. The model shows that besides interactions between the primer binding site and the last 10 nt at the 3'-end of tRNAi(Met), three short regions of Ty1 RNA named boxes 0, 1 and 2.1 interact with the T and D stems and loops of tRNAiMet. Mutations were made in the boxes or in the complementary sequences of tRNAi(Met) to study the contribution of these sequences to formation of the complex. We find that interaction with at least one of the two boxes 0 or 1 is absolutely required for efficient annealing of the two RNAs. Sequence comparison showing that the primary sequence of the boxes is strictly conserved in Ty1 and Ty2 elements and previously published in vivo results underline the functional importance of the primary sequence of the boxes and suggest that extended interactions between genomic Ty1 RNA and the primary tRNAi(Met) play a role in the reverse transcription pathway.},
note = {0305-1048
Journal Article},
keywords = {Acid, Base, cerevisiae, Conformation, Data, Gov't, Met/genetics/*metabolism, Molecular, Mutation, Non-U.S., Nucleic, Retroelements/*genetics, RNA, RNA/genetics/*metabolism, Saccharomyces, Sequence, structure, Support, Transfer},
pubstate = {published},
tppubtype = {article}
}
Reverse transcription of the yeast Ty1 retrotransposon is primed by tRNAi(Met) base paired to the primer binding site near the 5'-end of Ty1 genomic RNA. To understand the molecular basis of the tRNAi(Met)-Ty1 RNA interaction the secondary structure of the binary complex was analysed. Enzymatic probes were used to test the conformation of tRNAi(Met) and of Ty1 RNA in the free form and in the complex. A secondary structure model of the tRNAi(Met) Ty1 RNA complex consistent with the probing data was constructed with the help of a computer program. The model shows that besides interactions between the primer binding site and the last 10 nt at the 3'-end of tRNAi(Met), three short regions of Ty1 RNA named boxes 0, 1 and 2.1 interact with the T and D stems and loops of tRNAiMet. Mutations were made in the boxes or in the complementary sequences of tRNAi(Met) to study the contribution of these sequences to formation of the complex. We find that interaction with at least one of the two boxes 0 or 1 is absolutely required for efficient annealing of the two RNAs. Sequence comparison showing that the primary sequence of the boxes is strictly conserved in Ty1 and Ty2 elements and previously published in vivo results underline the functional importance of the primary sequence of the boxes and suggest that extended interactions between genomic Ty1 RNA and the primary tRNAi(Met) play a role in the reverse transcription pathway.
de Barros J. P. Pais, Keith G., Adlouni C. El, Glasser A. L., Mack G., Dirheimer G., Desgres J.
2'-O-methyl-5-formylcytidine (f5Cm), a new modified nucleotide at the 'wobble' of two cytoplasmic tRNAs Leu (NAA) from bovine liver Article de journal
Dans: Nucleic Acids Res, vol. 24, non 8, p. 1489-96, 1996, (0305-1048 Journal Article).
Résumé | BibTeX | Étiquettes: &, Acid, Acyl/*chemistry/isolation, Amino, Animals, Base, Borohydrides/chemistry, Cattle, Cells, Conformation, Cytidine/*analogs, Cytoplasm, Data, derivatives/chemistry/isolation, Fragmentography, Gov't, Hela, Human, Liver/*chemistry, Mass, Molecular, Non-U.S., Nucleic, purification, RNA, Sequence, structure, Support, Transfer
@article{,
title = {2'-O-methyl-5-formylcytidine (f5Cm), a new modified nucleotide at the 'wobble' of two cytoplasmic tRNAs Leu (NAA) from bovine liver},
author = { J. P. Pais de Barros and G. Keith and C. El Adlouni and A. L. Glasser and G. Mack and G. Dirheimer and J. Desgres},
year = {1996},
date = {1996-01-01},
journal = {Nucleic Acids Res},
volume = {24},
number = {8},
pages = {1489-96},
abstract = {The nucleotide analysis of a cytoplasmic tRNA(Leu) isolated from bovine liver revealed the presence of an unknown modified nucleotide N. The corresponding N nucleoside was isolated by different enzymatic and chromatographic protocols from a partially purified preparation of this tRNA(Leu). Its chemical characterization was determined from its chromatographic properties, UV-absorption spectroscopy and mass spectrometric measurements, as well as from those of the borohydride reduced N nucleoside and its etheno-trimethylsilyl derivative. The structure of N was established as 2'-O-methyl-5-formylcytidine (f5CM), and its reduced derivative as 2'-O-methyl-5-hydroxy-methylcytidine (om5Cm). By sequencing the bovine liver tRNA(Leu), the structure of the anticodon was determined as f5CmAA. In addition, the nucleotide sequence showed two primary structures differing only by the nucleotide 47c which is either uridine or adenosine. The two slightly differing bovine liver tRNAs-Leu(f5CmAA) are the only tRNAs so far sequenced which contain f5Cm. The role of such a modified cytidine at the first position of the anticodon is discussed in terms of decoding properties for the UUG and UUA leucine codons. Recently, precise evidence was obtained for the presence of f5Cm at the same position in tRNAs(Leu)(NAA) isolated from rabbit and lamb liver. Therefore, the 2'-O-methyl-5-formyl modification of cytidine at position 34 could be a general feature of cytoplasmic tRNAs(Leu)(NAA) in mammals.},
note = {0305-1048
Journal Article},
keywords = {&, Acid, Acyl/*chemistry/isolation, Amino, Animals, Base, Borohydrides/chemistry, Cattle, Cells, Conformation, Cytidine/*analogs, Cytoplasm, Data, derivatives/chemistry/isolation, Fragmentography, Gov't, Hela, Human, Liver/*chemistry, Mass, Molecular, Non-U.S., Nucleic, purification, RNA, Sequence, structure, Support, Transfer},
pubstate = {published},
tppubtype = {article}
}
The nucleotide analysis of a cytoplasmic tRNA(Leu) isolated from bovine liver revealed the presence of an unknown modified nucleotide N. The corresponding N nucleoside was isolated by different enzymatic and chromatographic protocols from a partially purified preparation of this tRNA(Leu). Its chemical characterization was determined from its chromatographic properties, UV-absorption spectroscopy and mass spectrometric measurements, as well as from those of the borohydride reduced N nucleoside and its etheno-trimethylsilyl derivative. The structure of N was established as 2'-O-methyl-5-formylcytidine (f5CM), and its reduced derivative as 2'-O-methyl-5-hydroxy-methylcytidine (om5Cm). By sequencing the bovine liver tRNA(Leu), the structure of the anticodon was determined as f5CmAA. In addition, the nucleotide sequence showed two primary structures differing only by the nucleotide 47c which is either uridine or adenosine. The two slightly differing bovine liver tRNAs-Leu(f5CmAA) are the only tRNAs so far sequenced which contain f5Cm. The role of such a modified cytidine at the first position of the anticodon is discussed in terms of decoding properties for the UUG and UUA leucine codons. Recently, precise evidence was obtained for the presence of f5Cm at the same position in tRNAs(Leu)(NAA) isolated from rabbit and lamb liver. Therefore, the 2'-O-methyl-5-formyl modification of cytidine at position 34 could be a general feature of cytoplasmic tRNAs(Leu)(NAA) in mammals.
1995
Gabryszuk J., Keith G., Monko M., Kuligowska E., Dirheimer G., Szarkowski J. W., Przykorska A.
Structural specificity of nuclease from wheat chloroplasts stroma Article de journal
Dans: Nucleic Acids Symp Ser, non 33, p. 115-9, 1995, (0261-3166 Journal Article).
Résumé | BibTeX | Étiquettes: &, Acid, Asp/chemistry/genetics/metabolism, Base, Binding, Chloroplasts/*enzymology, Conformation, Data, Endonucleases/isolation, Fungal/chemistry/genetics/metabolism, Gov't, Molecular, Non-U.S., Nucleic, Phe/chemistry/genetics/metabolism, purification/*metabolism, RNA, RNA/chemistry/metabolism, Sequence, Sites, Specificity, Substrate, Support, Transfer, Triticum/*enzymology
@article{,
title = {Structural specificity of nuclease from wheat chloroplasts stroma},
author = { J. Gabryszuk and G. Keith and M. Monko and E. Kuligowska and G. Dirheimer and J. W. Szarkowski and A. Przykorska},
year = {1995},
date = {1995-01-01},
journal = {Nucleic Acids Symp Ser},
number = {33},
pages = {115-9},
abstract = {A single-strand-specific nuclease from wheat chloroplasts (ChS nuclease) was tested as a tool for RNA secondary and tertiary structure investigations, using yeast tRNA(Phe) and yeast tRNA(Asp) as models. In tRNA(Phe) the nuclease introduced main primary cleavages at positions U33, A35 and A36 in the anticodon-loop and G18 and G19 in the D-loop. In tRNA(Asp) the main primary cleavages occurred at positions U33, G34 and U35 in the anticodon-loop and the lower one at position C20:1 in the D-loop. No primary cleavages were observed within the double-stranded stems. Because ChS nuclease has (i) a low molecular weight, (ii) a wide pH range of action (5.0 to 7.5) (iii) no divalent cation requirement in the reaction mixture and (iv) can be obtained as a pure protein in rather large quantities it appeared to be a very good tool for secondary and tertiary structural studies of RNAs.},
note = {0261-3166
Journal Article},
keywords = {&, Acid, Asp/chemistry/genetics/metabolism, Base, Binding, Chloroplasts/*enzymology, Conformation, Data, Endonucleases/isolation, Fungal/chemistry/genetics/metabolism, Gov't, Molecular, Non-U.S., Nucleic, Phe/chemistry/genetics/metabolism, purification/*metabolism, RNA, RNA/chemistry/metabolism, Sequence, Sites, Specificity, Substrate, Support, Transfer, Triticum/*enzymology},
pubstate = {published},
tppubtype = {article}
}
A single-strand-specific nuclease from wheat chloroplasts (ChS nuclease) was tested as a tool for RNA secondary and tertiary structure investigations, using yeast tRNA(Phe) and yeast tRNA(Asp) as models. In tRNA(Phe) the nuclease introduced main primary cleavages at positions U33, A35 and A36 in the anticodon-loop and G18 and G19 in the D-loop. In tRNA(Asp) the main primary cleavages occurred at positions U33, G34 and U35 in the anticodon-loop and the lower one at position C20:1 in the D-loop. No primary cleavages were observed within the double-stranded stems. Because ChS nuclease has (i) a low molecular weight, (ii) a wide pH range of action (5.0 to 7.5) (iii) no divalent cation requirement in the reaction mixture and (iv) can be obtained as a pure protein in rather large quantities it appeared to be a very good tool for secondary and tertiary structural studies of RNAs.
Heyman T., Agoutin B., Friant S., Wilhelm F. X., Wilhelm M. L.
Plus-strand DNA synthesis of the yeast retrotransposon Ty1 is initiated at two sites, PPT1 next to the 3' LTR and PPT2 within the pol gene. PPT1 is sufficient for Ty1 transposition Article de journal
Dans: J Mol Biol, vol. 253, non 2, p. 291-303, 1995, (0022-2836 Journal Article).
Résumé | BibTeX | Étiquettes: *DNA, *Genes, *Repetitive, *Retroelements, Acid, Base, C/analysis, cerevisiae/genetics/*virology, Chain, Cloning, Data, DNA, Fungal, Fungal/biosynthesis, Genes, Genetic, Genome, Gov't, Mapping, Molecular, Non-U.S., Nucleic, pol, Poly, Polymerase, Primers, Reaction, Replication, Restriction, Saccharomyces, Sequence, Sequences, Support, Transcription, Viral, Viral/*biosynthesis
@article{,
title = {Plus-strand DNA synthesis of the yeast retrotransposon Ty1 is initiated at two sites, PPT1 next to the 3' LTR and PPT2 within the pol gene. PPT1 is sufficient for Ty1 transposition},
author = { T. Heyman and B. Agoutin and S. Friant and F. X. Wilhelm and M. L. Wilhelm},
year = {1995},
date = {1995-01-01},
journal = {J Mol Biol},
volume = {253},
number = {2},
pages = {291-303},
abstract = {Long terminal repeat elements and retroviruses require primers for initiation of minus and plus-strand DNA synthesis by reverse transcriptase. Here we demonstrate genetically that plus-strand DNA synthesis of the yeast Ty1 element is initiated at two sites located at the 5' boundary of the 3' long terminal repeat (PPT1) and near the middle of the pol gene in the integrase coding sequence (PPT2). A consequence of the presence of two PPTs is that Ty1 plus-strand DNA exists as segments at some time during replication. Three fragments have been identified: the plus-strand strong-stop DNA initiated at PPT1, a downstream fragment initiated at PPT2 and an upstream fragment spanning the 5'-terminal part of Ty1 and a portion of the TyB gene. Characterization of the 3' ends of the plus-strand DNA fragments reveals (1) that the upstream fragment is elongated beyond PPT2 creating a plus-strand overlap and (2) that the majority of plus-strand strong-stop DNA fragments bear a copy of the minus-strand primer binding site in agreement with the accepted model of retroviral genomic RNA reverse transcription. The two polypurine tracts, PPT1 and PPT2, have an identical sequence GGGTGGTA. Mutations replacing purines by pyrimidines in this sequence significantly diminish or abolish initiation of plus-strand synthesis. Ty1 elements bearing a mutated PPT2 sequence are not defective for transposition whereas mutations in PPT1 abolish transposition.},
note = {0022-2836
Journal Article},
keywords = {*DNA, *Genes, *Repetitive, *Retroelements, Acid, Base, C/analysis, cerevisiae/genetics/*virology, Chain, Cloning, Data, DNA, Fungal, Fungal/biosynthesis, Genes, Genetic, Genome, Gov't, Mapping, Molecular, Non-U.S., Nucleic, pol, Poly, Polymerase, Primers, Reaction, Replication, Restriction, Saccharomyces, Sequence, Sequences, Support, Transcription, Viral, Viral/*biosynthesis},
pubstate = {published},
tppubtype = {article}
}
Long terminal repeat elements and retroviruses require primers for initiation of minus and plus-strand DNA synthesis by reverse transcriptase. Here we demonstrate genetically that plus-strand DNA synthesis of the yeast Ty1 element is initiated at two sites located at the 5' boundary of the 3' long terminal repeat (PPT1) and near the middle of the pol gene in the integrase coding sequence (PPT2). A consequence of the presence of two PPTs is that Ty1 plus-strand DNA exists as segments at some time during replication. Three fragments have been identified: the plus-strand strong-stop DNA initiated at PPT1, a downstream fragment initiated at PPT2 and an upstream fragment spanning the 5'-terminal part of Ty1 and a portion of the TyB gene. Characterization of the 3' ends of the plus-strand DNA fragments reveals (1) that the upstream fragment is elongated beyond PPT2 creating a plus-strand overlap and (2) that the majority of plus-strand strong-stop DNA fragments bear a copy of the minus-strand primer binding site in agreement with the accepted model of retroviral genomic RNA reverse transcription. The two polypurine tracts, PPT1 and PPT2, have an identical sequence GGGTGGTA. Mutations replacing purines by pyrimidines in this sequence significantly diminish or abolish initiation of plus-strand synthesis. Ty1 elements bearing a mutated PPT2 sequence are not defective for transposition whereas mutations in PPT1 abolish transposition.
O'Connor M., Brunelli C. A., Firpo M. A., Gregory S. T., Lieberman K. R., Lodmell J. S., Moine H., Ryk D. I. Van, Dahlberg A. E.
Genetic probes of ribosomal RNA function Article de journal
Dans: Biochem Cell Biol, vol. 73, non 11-12, p. 859-68, 1995, (0829-8211 Journal Article Review Review, Tutorial).
Résumé | BibTeX | Étiquettes: 16S/genetics, Acid, Base, Conformation, Data, Gov't, Messenger/genetics, Molecular, Mutation, Non-U.S., Nucleic, P.H.S., Probes, Ribosomal, Ribosomal/*genetics, RNA, Sequence, Support, Transfer/genetics, U.S.
@article{,
title = {Genetic probes of ribosomal RNA function},
author = { M. O'Connor and C. A. Brunelli and M. A. Firpo and S. T. Gregory and K. R. Lieberman and J. S. Lodmell and H. Moine and D. I. Van Ryk and A. E. Dahlberg},
year = {1995},
date = {1995-01-01},
journal = {Biochem Cell Biol},
volume = {73},
number = {11-12},
pages = {859-68},
abstract = {We have used a genetic approach to uncover the functional roles of rRNA in protein synthesis. Mutations were constructed in a cloned rrn operon by site-directed mutagenesis or isolated by genetic selections following random mutagenesis. We have identified mutations that affect each step in the process of translation. The data are consistent with the results of biochemical and phylogenetic analyses but, in addition, have provided novel information on regions of rRNA not previously investigated.},
note = {0829-8211
Journal Article
Review
Review, Tutorial},
keywords = {16S/genetics, Acid, Base, Conformation, Data, Gov't, Messenger/genetics, Molecular, Mutation, Non-U.S., Nucleic, P.H.S., Probes, Ribosomal, Ribosomal/*genetics, RNA, Sequence, Support, Transfer/genetics, U.S.},
pubstate = {published},
tppubtype = {article}
}
We have used a genetic approach to uncover the functional roles of rRNA in protein synthesis. Mutations were constructed in a cloned rrn operon by site-directed mutagenesis or isolated by genetic selections following random mutagenesis. We have identified mutations that affect each step in the process of translation. The data are consistent with the results of biochemical and phylogenetic analyses but, in addition, have provided novel information on regions of rRNA not previously investigated.
1994
Heyman T., Agoutin B., Fix C., Dirheimer G., Keith G.
Yeast serine isoacceptor tRNAs: variations of their content as a function of growth conditions and primary structure of the minor tRNA(Ser)GCU Article de journal
Dans: FEBS Lett, vol. 347, non 2-3, p. 143-6, 1994, (0014-5793 Journal Article).
Résumé | BibTeX | Étiquettes: &, Acid, Anticodon, Base, cerevisiae/*genetics/*growth, Conformation, Culture, Data, development, Fungal/*chemistry, Galactose, Hybridization, Media, Molecular, Nucleic, Probes, RNA, Saccharomyces, Sequence, Ser/analysis/*chemistry, Transfer, Transfer/*chemistry
@article{,
title = {Yeast serine isoacceptor tRNAs: variations of their content as a function of growth conditions and primary structure of the minor tRNA(Ser)GCU},
author = { T. Heyman and B. Agoutin and C. Fix and G. Dirheimer and G. Keith},
year = {1994},
date = {1994-01-01},
journal = {FEBS Lett},
volume = {347},
number = {2-3},
pages = {143-6},
abstract = {The primary structure of Saccharomyces cerevisiae tRNA(Ser)GCU is presented (EMBL database accession No. X74268 S. cerevisiae tRNA-Ser). In addition, quantitation of the relative amounts of serine isoaccepting tRNAs in yeast grown on different media showed that the minor tRNA(Ser)GCU decreased while the major tRNA(Ser)AGA increased as the growth rate and the cellular protein content increased. The minor species, tRNA(Ser)CGA and tRNA(Ser)UGA, were not separated by our gel system, however, taken together they appeared to vary in the same way as tRNA(Ser)GCU. These data suggest a growth rate dependence of yeast tRNAs similar to that previously described for E. coli tRNAs.},
note = {0014-5793
Journal Article},
keywords = {&, Acid, Anticodon, Base, cerevisiae/*genetics/*growth, Conformation, Culture, Data, development, Fungal/*chemistry, Galactose, Hybridization, Media, Molecular, Nucleic, Probes, RNA, Saccharomyces, Sequence, Ser/analysis/*chemistry, Transfer, Transfer/*chemistry},
pubstate = {published},
tppubtype = {article}
}
The primary structure of Saccharomyces cerevisiae tRNA(Ser)GCU is presented (EMBL database accession No. X74268 S. cerevisiae tRNA-Ser). In addition, quantitation of the relative amounts of serine isoaccepting tRNAs in yeast grown on different media showed that the minor tRNA(Ser)GCU decreased while the major tRNA(Ser)AGA increased as the growth rate and the cellular protein content increased. The minor species, tRNA(Ser)CGA and tRNA(Ser)UGA, were not separated by our gel system, however, taken together they appeared to vary in the same way as tRNA(Ser)GCU. These data suggest a growth rate dependence of yeast tRNAs similar to that previously described for E. coli tRNAs.
1993
el Adlouni C., Keith G., Dirheimer G., Szarkowski J. W., Przykorska A.
Rye nuclease I as a tool for structural studies of tRNAs with large variable arms Article de journal
Dans: Nucleic Acids Res, vol. 21, non 4, p. 941-7, 1993, (0305-1048 Journal Article).
Résumé | BibTeX | Étiquettes: *Nucleotidases, Acid, Animals, Anticodon, Base, Cattle, cereale/*enzymology, cerevisiae, Conformation, Data, Gov't, Leu/chemistry, Molecular, Non-U.S., Nucleic, RNA, Saccharomyces, Secale, Sequence, Ser/chemistry, Support, Transfer, Transfer/*chemistry
@article{,
title = {Rye nuclease I as a tool for structural studies of tRNAs with large variable arms},
author = { C. el Adlouni and G. Keith and G. Dirheimer and J. W. Szarkowski and A. Przykorska},
year = {1993},
date = {1993-01-01},
journal = {Nucleic Acids Res},
volume = {21},
number = {4},
pages = {941-7},
abstract = {A single-strand-specific nuclease from rye germ (Rn nuclease I) was used for secondary and tertiary structure investigations of tRNAs with large variable arms (class II tRNAs). We have studied the structure in solution of two recently sequenced tRNA(Leu): yeast tRNA(Leu)(ncm5UmAA) and bovine tRNA(Leu)(XmAA) as well as yeast tRNA(Leu)(UAG), tRNA(Leu)(m5CAA) and tRNA(Ser)(IGA). The latter is the only tRNA with a long variable arm for which the secondary and tertiary structure has already been studied by use of chemical probes and computer modelling. The data obtained in this work showed that the general model of class II tRNAs proposed by others for tRNA(Ser) can be extended to tRNAs(Leu) as well. However interesting differences in the structure of tRNAs(Leu) versus tRNA(Ser)(IGA) were also noticed. The main difference was observed in the accessibility of the variable loops to nucleolytic attack of Rn nuclease I: variable loops of all studied tRNA(Leu) species were cut by Rn nuclease I, while that of yeast tRNA(Ser)(IGA) was not. This could be due to differences in stability of the variable arms and the lengths of their loops which are 3 and 4 nucleotides in tRNA(Ser)(IGA) and tRNAs(Leu) respectively.},
note = {0305-1048
Journal Article},
keywords = {*Nucleotidases, Acid, Animals, Anticodon, Base, Cattle, cereale/*enzymology, cerevisiae, Conformation, Data, Gov't, Leu/chemistry, Molecular, Non-U.S., Nucleic, RNA, Saccharomyces, Secale, Sequence, Ser/chemistry, Support, Transfer, Transfer/*chemistry},
pubstate = {published},
tppubtype = {article}
}
A single-strand-specific nuclease from rye germ (Rn nuclease I) was used for secondary and tertiary structure investigations of tRNAs with large variable arms (class II tRNAs). We have studied the structure in solution of two recently sequenced tRNA(Leu): yeast tRNA(Leu)(ncm5UmAA) and bovine tRNA(Leu)(XmAA) as well as yeast tRNA(Leu)(UAG), tRNA(Leu)(m5CAA) and tRNA(Ser)(IGA). The latter is the only tRNA with a long variable arm for which the secondary and tertiary structure has already been studied by use of chemical probes and computer modelling. The data obtained in this work showed that the general model of class II tRNAs proposed by others for tRNA(Ser) can be extended to tRNAs(Leu) as well. However interesting differences in the structure of tRNAs(Leu) versus tRNA(Ser)(IGA) were also noticed. The main difference was observed in the accessibility of the variable loops to nucleolytic attack of Rn nuclease I: variable loops of all studied tRNA(Leu) species were cut by Rn nuclease I, while that of yeast tRNA(Ser)(IGA) was not. This could be due to differences in stability of the variable arms and the lengths of their loops which are 3 and 4 nucleotides in tRNA(Ser)(IGA) and tRNAs(Leu) respectively.
Pochart P., Agoutin B., Fix C., Keith G., Heyman T.
A very poorly expressed tRNA(Ser) is highly concentrated together with replication primer initiator tRNA(Met) in the yeast Ty1 virus-like particles Article de journal
Dans: Nucleic Acids Res, vol. 21, non 7, p. 1517-21, 1993, (0305-1048 Journal Article).
Résumé | BibTeX | Étiquettes: &, Acid, Base, cerevisiae/metabolism, Conformation, Data, development, DNA, Electrophoresis, Elements/*physiology, Gel, Met/metabolism, Molecular, Nucleic, Retroviridae/*growth, RNA, Saccharomyces, Sequence, Ser/*metabolism, Transfer, Transposable, Two-Dimensional, Viral/*metabolism
@article{,
title = {A very poorly expressed tRNA(Ser) is highly concentrated together with replication primer initiator tRNA(Met) in the yeast Ty1 virus-like particles},
author = { P. Pochart and B. Agoutin and C. Fix and G. Keith and T. Heyman},
year = {1993},
date = {1993-01-01},
journal = {Nucleic Acids Res},
volume = {21},
number = {7},
pages = {1517-21},
abstract = {The analysis of the tRNAs associated to the virus-like particles produced by the Ty1 element revealed the specific packaging of three major tRNA species, in about equal amounts: the replication primer initiator tRNA(Met), the tRNA(Ser)AGA and a tRNA undetected until now as an expressed species in yeast. The latter tRNA is coded by the already described tDNA(Ser)GCT. This tRNA is enriched more than 150 fold in the particles as compared to its content in total cellular tRNA where it represents less than 0.1% (initiator tRNA(Met) and tRNA(Ser)AGA being 11 and 4 fold enriched respectively). This tRNA is the only species coded by the tDNA(Ser)GCT gene which is found in three copies per genome since no other corresponding expressed tRNA could be detected. This gene is thus very poorly expressed. The high concentration of tRNA(Ser)GCU in the particles compared to its very low cellular content led us to consider its possible implication in Ty specific processes.},
note = {0305-1048
Journal Article},
keywords = {&, Acid, Base, cerevisiae/metabolism, Conformation, Data, development, DNA, Electrophoresis, Elements/*physiology, Gel, Met/metabolism, Molecular, Nucleic, Retroviridae/*growth, RNA, Saccharomyces, Sequence, Ser/*metabolism, Transfer, Transposable, Two-Dimensional, Viral/*metabolism},
pubstate = {published},
tppubtype = {article}
}
The analysis of the tRNAs associated to the virus-like particles produced by the Ty1 element revealed the specific packaging of three major tRNA species, in about equal amounts: the replication primer initiator tRNA(Met), the tRNA(Ser)AGA and a tRNA undetected until now as an expressed species in yeast. The latter tRNA is coded by the already described tDNA(Ser)GCT. This tRNA is enriched more than 150 fold in the particles as compared to its content in total cellular tRNA where it represents less than 0.1% (initiator tRNA(Met) and tRNA(Ser)AGA being 11 and 4 fold enriched respectively). This tRNA is the only species coded by the tDNA(Ser)GCT gene which is found in three copies per genome since no other corresponding expressed tRNA could be detected. This gene is thus very poorly expressed. The high concentration of tRNA(Ser)GCU in the particles compared to its very low cellular content led us to consider its possible implication in Ty specific processes.
Santos M. A., Keith G., Tuite M. F.
Non-standard translational events in Candida albicans mediated by an unusual seryl-tRNA with a 5'-CAG-3' (leucine) anticodon Article de journal
Dans: EMBO J, vol. 12, non 2, p. 607-16, 1993, (0261-4189 Journal Article).
Résumé | BibTeX | Étiquettes: *Anticodon, *Translation, &, Acid, albicans/*genetics, Base, Candida, Cloning, Conformation, Data, DNA, Fungal, Fungal/chemistry/genetics/isolation, Genes, Genetic, Gov't, Leucine/*genetics, Molecular, Non-U.S., Nucleic, purification, RNA, Sequence, Ser/chemistry/*genetics/isolation, Support, Transfer
@article{,
title = {Non-standard translational events in Candida albicans mediated by an unusual seryl-tRNA with a 5'-CAG-3' (leucine) anticodon},
author = { M. A. Santos and G. Keith and M. F. Tuite},
year = {1993},
date = {1993-01-01},
journal = {EMBO J},
volume = {12},
number = {2},
pages = {607-16},
abstract = {From in vitro translation studies we have previously demonstrated the existence of an apparent efficient UAG (amber) suppressor tRNA in the dimorphic fungus Candida albicans (Santos et al., 1990). Using an in vitro assay for termination codon readthrough the tRNA responsible was purified to homogeneity from C.albicans cells. The determined sequence of the purified tRNA predicts a 5'-CAG-3' anticodon that should decode the leucine codon CUG and not the UAG termination codon as originally hypothesized. However, the tRNA(CAG) sequence shows greater nucleotide homology with seryl-tRNAs from the closely related yeast Saccharomyces cerevisiae than with leucyl-tRNAs from the same species. In vitro tRNA-charging studies demonstrated that the purified tRNA(CAG) is charged with Ser. The gene encoding the tRNA was cloned from C.albicans by a PCR-based strategy and DNA sequence analysis confirmed both the structure of the tRNA(CAG) and the absence of any introns in the tRNA gene. The copy number of the tRNA(CAG) gene (1-2 genes per haploid genome) is in agreement with the relatively low abundance (< 0.5% total tRNA) of this tRNA. In vitro translation studies revealed that the purified tRNA(CAG) could induce apparent translational bypass of all three termination codons. However, peptide mapping of in vitro translation products demonstrated that the tRNA(CAG) induces translational misreading in the amino-terminal region of two RNA templates employed, namely the rabbit alpha- and beta-globin mRNAs. These results suggest that the C.albicans tRNA(CAG) is not an 'omnipotent' suppressor tRNA but rather may mediate a novel non-standard translational event in vitro during the translation of the CUG codon. The possible nature of this non-standard translation event is discussed in the context of both the unusual structural features of the tRNA(CAG) and its in vitro behaviour.},
note = {0261-4189
Journal Article},
keywords = {*Anticodon, *Translation, &, Acid, albicans/*genetics, Base, Candida, Cloning, Conformation, Data, DNA, Fungal, Fungal/chemistry/genetics/isolation, Genes, Genetic, Gov't, Leucine/*genetics, Molecular, Non-U.S., Nucleic, purification, RNA, Sequence, Ser/chemistry/*genetics/isolation, Support, Transfer},
pubstate = {published},
tppubtype = {article}
}
From in vitro translation studies we have previously demonstrated the existence of an apparent efficient UAG (amber) suppressor tRNA in the dimorphic fungus Candida albicans (Santos et al., 1990). Using an in vitro assay for termination codon readthrough the tRNA responsible was purified to homogeneity from C.albicans cells. The determined sequence of the purified tRNA predicts a 5'-CAG-3' anticodon that should decode the leucine codon CUG and not the UAG termination codon as originally hypothesized. However, the tRNA(CAG) sequence shows greater nucleotide homology with seryl-tRNAs from the closely related yeast Saccharomyces cerevisiae than with leucyl-tRNAs from the same species. In vitro tRNA-charging studies demonstrated that the purified tRNA(CAG) is charged with Ser. The gene encoding the tRNA was cloned from C.albicans by a PCR-based strategy and DNA sequence analysis confirmed both the structure of the tRNA(CAG) and the absence of any introns in the tRNA gene. The copy number of the tRNA(CAG) gene (1-2 genes per haploid genome) is in agreement with the relatively low abundance (< 0.5% total tRNA) of this tRNA. In vitro translation studies revealed that the purified tRNA(CAG) could induce apparent translational bypass of all three termination codons. However, peptide mapping of in vitro translation products demonstrated that the tRNA(CAG) induces translational misreading in the amino-terminal region of two RNA templates employed, namely the rabbit alpha- and beta-globin mRNAs. These results suggest that the C.albicans tRNA(CAG) is not an 'omnipotent' suppressor tRNA but rather may mediate a novel non-standard translational event in vitro during the translation of the CUG codon. The possible nature of this non-standard translation event is discussed in the context of both the unusual structural features of the tRNA(CAG) and its in vitro behaviour.
1992
Heitzler J., Marechal-Drouard L., Dirheimer G., Keith G.
Use of a dot blot hybridization method for identification of pure tRNA species on different membranes Article de journal
Dans: Biochim Biophys Acta-Gene Regul Mech, vol. 1129, non 3, p. 273-7, 1992, (0006-3002 Journal Article).
Résumé | BibTeX | Étiquettes: *Membranes, Acid, Artificial, Autoradiography, cerevisiae/genetics, Fungal/genetics, Gov't, Hybridization, Met/genetics, Non-U.S., Nucleic, RNA, Saccharomyces, Support, Transfer, Transfer/*genetics
@article{,
title = {Use of a dot blot hybridization method for identification of pure tRNA species on different membranes},
author = { J. Heitzler and L. Marechal-Drouard and G. Dirheimer and G. Keith},
year = {1992},
date = {1992-01-01},
journal = {Biochim Biophys Acta-Gene Regul Mech},
volume = {1129},
number = {3},
pages = {273-7},
abstract = {The characterization of a tRNA in purification procedures usually involves aminoacylation assays but recently, the hybridization by dot blot with specific oligonucleotides as probes has been used for the tRNA identification. We present here an optimization of a dot blot hybridization method for the tRNA detection by comparing the efficiency of eight different nylon membranes. Neutral 0.22 microns porosity membranes (Nytran, Biodine A) give the best detection efficiency when small quantities of material (less than 40 ng of tRNA) are dotted on filter; by contrast, neutral 0.45 microns porosity membranes (such as Hybond N) are the most efficient when larger quantities of tRNA are dotted on the filter. The described technique allows to detect less than 20 pg of a pure tRNA species. Its use in the identification of Saccharomyces cerevisiae initiator tRNA(Met) in counter-current distribution fractions is shown.},
note = {0006-3002
Journal Article},
keywords = {*Membranes, Acid, Artificial, Autoradiography, cerevisiae/genetics, Fungal/genetics, Gov't, Hybridization, Met/genetics, Non-U.S., Nucleic, RNA, Saccharomyces, Support, Transfer, Transfer/*genetics},
pubstate = {published},
tppubtype = {article}
}
The characterization of a tRNA in purification procedures usually involves aminoacylation assays but recently, the hybridization by dot blot with specific oligonucleotides as probes has been used for the tRNA identification. We present here an optimization of a dot blot hybridization method for the tRNA detection by comparing the efficiency of eight different nylon membranes. Neutral 0.22 microns porosity membranes (Nytran, Biodine A) give the best detection efficiency when small quantities of material (less than 40 ng of tRNA) are dotted on filter; by contrast, neutral 0.45 microns porosity membranes (such as Hybond N) are the most efficient when larger quantities of tRNA are dotted on the filter. The described technique allows to detect less than 20 pg of a pure tRNA species. Its use in the identification of Saccharomyces cerevisiae initiator tRNA(Met) in counter-current distribution fractions is shown.
Przykorska A., el Adlouni C., Keith G., Szarkowski J. W., Dirheimer G.
Structural specificity of Rn nuclease I as probed on yeast tRNA(Phe) and tRNA(Asp) Article de journal
Dans: Nucleic Acids Res, vol. 20, non 4, p. 659-63, 1992, (0305-1048 Journal Article).
Résumé | BibTeX | Étiquettes: Acid, Asp/chemistry/genetics/*metabolism, Base, cereale, Composition, Conformation, Data, Gov't, Molecular, Non-U.S., Nucleic, Pancreatic/*metabolism, Phe/chemistry/genetics/*metabolism, Ribonuclease, RNA, Secale, Sequence, Specificity, Substrate, Support, Transfer, Yeasts/genetics
@article{,
title = {Structural specificity of Rn nuclease I as probed on yeast tRNA(Phe) and tRNA(Asp)},
author = { A. Przykorska and C. el Adlouni and G. Keith and J. W. Szarkowski and G. Dirheimer},
year = {1992},
date = {1992-01-01},
journal = {Nucleic Acids Res},
volume = {20},
number = {4},
pages = {659-63},
abstract = {A single-strand-specific nuclease from rye germ (Rn nuclease I) was characterized as a tool for secondary and tertiary structure investigation of RNAs. To test the procedure, yeast tRNA(Phe) and tRNA(Asp) for which the tertiary structures are known, as well as the 3'-half of tRNA(Asp) were used as substrates. In tRNA(Phe) the nuclease introduced main primary cuts at positions U33 and A35 of the anticodon loop and G18 and G19 of the D loop. No primary cuts were observed within the double stranded stems. In tRNA(Asp) the main cuts occurred at positions U33, G34, U35, C36 of the anticodon loop and G18 and C20:1 positions in the D loop. No cuts were observed in the T loop in intact tRNA(Asp) but strong primary cleavages occurred at positions psi 55, C56, A57 within that loop in the absence of the tertiary interactions between T and D loops (use of 3'-half tRNA(Asp)). These results show that Rn nuclease I is specific for exposed single-stranded regions.},
note = {0305-1048
Journal Article},
keywords = {Acid, Asp/chemistry/genetics/*metabolism, Base, cereale, Composition, Conformation, Data, Gov't, Molecular, Non-U.S., Nucleic, Pancreatic/*metabolism, Phe/chemistry/genetics/*metabolism, Ribonuclease, RNA, Secale, Sequence, Specificity, Substrate, Support, Transfer, Yeasts/genetics},
pubstate = {published},
tppubtype = {article}
}
A single-strand-specific nuclease from rye germ (Rn nuclease I) was characterized as a tool for secondary and tertiary structure investigation of RNAs. To test the procedure, yeast tRNA(Phe) and tRNA(Asp) for which the tertiary structures are known, as well as the 3'-half of tRNA(Asp) were used as substrates. In tRNA(Phe) the nuclease introduced main primary cuts at positions U33 and A35 of the anticodon loop and G18 and G19 of the D loop. No primary cuts were observed within the double stranded stems. In tRNA(Asp) the main cuts occurred at positions U33, G34, U35, C36 of the anticodon loop and G18 and C20:1 positions in the D loop. No cuts were observed in the T loop in intact tRNA(Asp) but strong primary cleavages occurred at positions psi 55, C56, A57 within that loop in the absence of the tertiary interactions between T and D loops (use of 3'-half tRNA(Asp)). These results show that Rn nuclease I is specific for exposed single-stranded regions.