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1992
Perret V, Florentz C, Puglisi J D, Giege R
Effect of conformational features on the aminoacylation of tRNAs and consequences on the permutation of tRNA specificities Journal Article
In: J Mol Biol, vol. 226, no. 2, pp. 323-333, 1992, ISBN: 1640453, (0022-2836 Journal Article).
Abstract | Links | BibTeX | Tags: Amino Acid Activation Aspartate-tRNA Ligase/*metabolism Base Sequence Molecular Sequence Data Nucleic Acid Conformation Phenylalanine-tRNA Ligase/*metabolism RNA, Asp/metabolism/*ultrastructure RNA, FLORENTZ, Fungal/metabolism/ultrastructure RNA, Non-U.S. Gov't, Phe/metabolism/*ultrastructure Saccharomyces cerevisiae Structure-Activity Relationship Substrate Specificity Support, Transfer, Unité ARN
@article{,
title = {Effect of conformational features on the aminoacylation of tRNAs and consequences on the permutation of tRNA specificities},
author = {V Perret and C Florentz and J D Puglisi and R Giege},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1640453},
isbn = {1640453},
year = {1992},
date = {1992-01-01},
journal = {J Mol Biol},
volume = {226},
number = {2},
pages = {323-333},
abstract = {The structure and function of in vitro transcribed tRNA(Asp) variants with inserted conformational features characteristic of yeast tRNA(Phe), such as the length of the variable region or the arrangement of the conserved residues in the D-loop, have been investigated. Although they exhibit significant conformational alterations as revealed by Pb2+ treatment, these variants are still efficiently aspartylated by yeast aspartyl-tRNA synthetase. Thus, this synthetase can accommodate a variety of tRNA conformers. In a second series of variants, the identity determinants of yeast tRNA(Phe) were transplanted into the previous structural variants of tRNA(Asp). The phenylalanine acceptance of these variants improves with increasing the number of structural characteristics of tRNA(Phe), suggesting that phenylalanyl-tRNA synthetase is sensitive to the conformational frame embedding the cognate identity nucleotides. These results contrast with the efficient transplantation of tRNA(Asp) identity elements into yeast tRNA(Phe). This indicates that synthetases respond differently to the detailed conformation of their tRNA substrates. Efficient aminoacylation is not only dependent on the presence of the set of identity nucleotides, but also on a precise conformation of the tRNA.},
note = {0022-2836
Journal Article},
keywords = {Amino Acid Activation Aspartate-tRNA Ligase/*metabolism Base Sequence Molecular Sequence Data Nucleic Acid Conformation Phenylalanine-tRNA Ligase/*metabolism RNA, Asp/metabolism/*ultrastructure RNA, FLORENTZ, Fungal/metabolism/ultrastructure RNA, Non-U.S. Gov't, Phe/metabolism/*ultrastructure Saccharomyces cerevisiae Structure-Activity Relationship Substrate Specificity Support, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The structure and function of in vitro transcribed tRNA(Asp) variants with inserted conformational features characteristic of yeast tRNA(Phe), such as the length of the variable region or the arrangement of the conserved residues in the D-loop, have been investigated. Although they exhibit significant conformational alterations as revealed by Pb2+ treatment, these variants are still efficiently aspartylated by yeast aspartyl-tRNA synthetase. Thus, this synthetase can accommodate a variety of tRNA conformers. In a second series of variants, the identity determinants of yeast tRNA(Phe) were transplanted into the previous structural variants of tRNA(Asp). The phenylalanine acceptance of these variants improves with increasing the number of structural characteristics of tRNA(Phe), suggesting that phenylalanyl-tRNA synthetase is sensitive to the conformational frame embedding the cognate identity nucleotides. These results contrast with the efficient transplantation of tRNA(Asp) identity elements into yeast tRNA(Phe). This indicates that synthetases respond differently to the detailed conformation of their tRNA substrates. Efficient aminoacylation is not only dependent on the presence of the set of identity nucleotides, but also on a precise conformation of the tRNA.
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 Journal Article
In: Biochim Biophys Acta-Gene Regul Mech, vol. 1129, no. 3, pp. 273-277, 1992, ISBN: 1536878, (0006-3002 Journal Article).
Abstract | Links | BibTeX | Tags: Artificial Nucleic Acid Hybridization RNA, Autoradiography *Membranes, Fungal/genetics RNA, Met/genetics Saccharomyces cerevisiae/genetics Support, Non-U.S. Gov't, Transfer, Transfer/*genetics RNA, Unité ARN
@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},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1536878},
isbn = {1536878},
year = {1992},
date = {1992-01-01},
journal = {Biochim Biophys Acta-Gene Regul Mech},
volume = {1129},
number = {3},
pages = {273-277},
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 = {Artificial Nucleic Acid Hybridization RNA, Autoradiography *Membranes, Fungal/genetics RNA, Met/genetics Saccharomyces cerevisiae/genetics Support, Non-U.S. Gov't, Transfer, Transfer/*genetics RNA, Unité ARN},
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.
Garcia A, Giege R
Footprinting evidence for close contacts of the yeast tRNA(Asp) anticodon region with aspartyl-tRNA synthetase Journal Article
In: Biochem Biophys Res Commun, vol. 186, no. 2, pp. 956-962, 1992, ISBN: 1497679, (0006-291x Journal Article).
Abstract | Links | BibTeX | Tags: Alkylation Anticodon/*metabolism Aspartate-tRNA Ligase/*metabolism Base Sequence Molecular Sequence Data Nucleic Acid Conformation Protein Binding RNA, Asp/genetics/isolation & purification/*metabolism Saccharomyces cerevisiae/enzymology/*genetics Sulfuric Acid Esters/metabolism/pharmacology Support, Non-U.S. Gov't, Transfer, Unité ARN
@article{,
title = {Footprinting evidence for close contacts of the yeast tRNA(Asp) anticodon region with aspartyl-tRNA synthetase},
author = {A Garcia and R Giege},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1497679},
isbn = {1497679},
year = {1992},
date = {1992-01-01},
journal = {Biochem Biophys Res Commun},
volume = {186},
number = {2},
pages = {956-962},
abstract = {Chemical footprinting experiments on brewer's yeast tRNA(Asp) complexed to its cognate aspartyl-tRNA synthetase are reported: they demonstrate that bases of the anticodon loop, including the anticodon itself, are in close proximity with the synthetase. Contacts were determined using dimethylsulfate as the probe for testing reactivity of guanine and cytosine residues in free and complexed tRNA. Results correlate with the decrease in aspartylation activity of yeast tRNA(Asp) molecules mutated at these contact positions and will be compared with other structural data arising from solution and crystallographic studies on the aspartic acid complex.},
note = {0006-291x
Journal Article},
keywords = {Alkylation Anticodon/*metabolism Aspartate-tRNA Ligase/*metabolism Base Sequence Molecular Sequence Data Nucleic Acid Conformation Protein Binding RNA, Asp/genetics/isolation & purification/*metabolism Saccharomyces cerevisiae/enzymology/*genetics Sulfuric Acid Esters/metabolism/pharmacology Support, Non-U.S. Gov't, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Chemical footprinting experiments on brewer's yeast tRNA(Asp) complexed to its cognate aspartyl-tRNA synthetase are reported: they demonstrate that bases of the anticodon loop, including the anticodon itself, are in close proximity with the synthetase. Contacts were determined using dimethylsulfate as the probe for testing reactivity of guanine and cytosine residues in free and complexed tRNA. Results correlate with the decrease in aspartylation activity of yeast tRNA(Asp) molecules mutated at these contact positions and will be compared with other structural data arising from solution and crystallographic studies on the aspartic acid complex.
Despons L, Senger B, Fasiolo F, Walter P
Binding of the yeast tRNA(Met) anticodon by the cognate methionyl-tRNA synthetase involves at least two independent peptide regions Journal Article
In: J Mol Biol, vol. 225, no. 3, pp. 897-907, 1992, ISBN: 1602489, (0022-2836 Journal Article).
Abstract | Links | BibTeX | Tags: Amino Acid Sequence Anticodon/*metabolism Binding Sites Kinetics Methionine-tRNA Ligase/*metabolism Models, Met/*metabolism Saccharomyces cerevisiae/enzymology Structure-Activity Relationship Support, Molecular Molecular Sequence Data Mutagenesis, Non-U.S. Gov't, Site-Directed Protein Conformation RNA, Transfer, Unité ARN
@article{,
title = {Binding of the yeast tRNA(Met) anticodon by the cognate methionyl-tRNA synthetase involves at least two independent peptide regions},
author = {L Despons and B Senger and F Fasiolo and P Walter},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1602489},
isbn = {1602489},
year = {1992},
date = {1992-01-01},
journal = {J Mol Biol},
volume = {225},
number = {3},
pages = {897-907},
abstract = {As for Escherichia coli methionine tRNAs, the anticodon triplet of yeast tRNA(Met) plays an important role in the recognition by the yeast methionyl-tRNA synthetase (MetRS), indicating that this determinant for methionine identity is conserved in yeast. Efficient aminoacylation of the E. coli tRNA(Met) transcript by the heterologous yeast methionine enzyme also suggests conservation of the protein determinants that interact with the CAU anticodon sequence. We have analysed by site-directed mutagenesis the peptide region 655 to 663 of the yeast MetRS that is equivalent to the anticodon binding region of the E. coli methionine enzyme. Only one change, converting Leu658 into Ala significantly reduced tRNA aminoacylation. Semi-conservative substitutions of L658 allow a correlation to be drawn between side-chain volume of the hydrophobic residue at this site and activity. The analysis of the L658A mutant shows that Km is mainly affected. This suggests that the peptide region 655 to 663 contributes partially to the binding of the anticodon, since separate mutational analysis of the anticodon bases shows that kcat is the most critical parameter in the recognition of tRNA(Met) by the yeast synthetase. We have analysed the role of peptide region (583-GNLVNR-588) that is spatially close to the region 655 to 663. Replacements of residues N584 and R588 reduces significantly the kcat of aminoacylation. The peptide region 583-GNLVNR-588 is highly conserved in all MetRS so far sequenced. We therefore propose that the hydrogen donor/acceptor amino acid residues within this region are the most critical protein determinants for the positive selection of the methionine tRNAs.},
note = {0022-2836
Journal Article},
keywords = {Amino Acid Sequence Anticodon/*metabolism Binding Sites Kinetics Methionine-tRNA Ligase/*metabolism Models, Met/*metabolism Saccharomyces cerevisiae/enzymology Structure-Activity Relationship Support, Molecular Molecular Sequence Data Mutagenesis, Non-U.S. Gov't, Site-Directed Protein Conformation RNA, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
As for Escherichia coli methionine tRNAs, the anticodon triplet of yeast tRNA(Met) plays an important role in the recognition by the yeast methionyl-tRNA synthetase (MetRS), indicating that this determinant for methionine identity is conserved in yeast. Efficient aminoacylation of the E. coli tRNA(Met) transcript by the heterologous yeast methionine enzyme also suggests conservation of the protein determinants that interact with the CAU anticodon sequence. We have analysed by site-directed mutagenesis the peptide region 655 to 663 of the yeast MetRS that is equivalent to the anticodon binding region of the E. coli methionine enzyme. Only one change, converting Leu658 into Ala significantly reduced tRNA aminoacylation. Semi-conservative substitutions of L658 allow a correlation to be drawn between side-chain volume of the hydrophobic residue at this site and activity. The analysis of the L658A mutant shows that Km is mainly affected. This suggests that the peptide region 655 to 663 contributes partially to the binding of the anticodon, since separate mutational analysis of the anticodon bases shows that kcat is the most critical parameter in the recognition of tRNA(Met) by the yeast synthetase. We have analysed the role of peptide region (583-GNLVNR-588) that is spatially close to the region 655 to 663. Replacements of residues N584 and R588 reduces significantly the kcat of aminoacylation. The peptide region 583-GNLVNR-588 is highly conserved in all MetRS so far sequenced. We therefore propose that the hydrogen donor/acceptor amino acid residues within this region are the most critical protein determinants for the positive selection of the methionine tRNAs.
