Publications
2003
Geslain R, Martin F, Camasses A, Eriani G
A yeast knockout strain to discriminate between active and inactive tRNA molecules Article de journal
Dans: Nucleic Acids Res, vol. 31, no. 16, p. 4729-4737, 2003, ISBN: 12907713, (1362-4962 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: Amino Acyl-tRNA Ligases/metabolism Arginine/genetics/metabolism Base Sequence Blotting, Arg/chemistry/*genetics/metabolism Saccharomyces cerevisiae/*genetics Support, ERIANI, Molecular Hydrogen-Ion Concentration Molecular Sequence Data Mutagenesis, Non-U.S. Gov't, Northern Cloning, Site-Directed Mutation Nucleic Acid Conformation RNA, Transfer, Unité ARN
@article{,
title = {A yeast knockout strain to discriminate between active and inactive tRNA molecules},
author = {R Geslain and F Martin and A Camasses and G Eriani},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12907713},
isbn = {12907713},
year = {2003},
date = {2003-01-01},
journal = {Nucleic Acids Res},
volume = {31},
number = {16},
pages = {4729-4737},
abstract = {Here we report the construction of a yeast genetic screen designed to identify essential residues in tRNA(Arg). The system consists of a tRNA(Arg) knockout strain and a set of vectors designed to rescue and select for variants of tRNA(Arg). By plasmid shuffling we selected inactive tRNA mutants that were further analyzed by northern blotting. The mutational analysis focused on the tRNA D and anticodon loops that contact the aminoacyl-tRNA synthetase. The anticodon triplet was excluded from the analysis because of its role in decoding the Arg codons. Most of the inactivating mutations are residues involved in tertiary interactions. These mutations had dramatic effects on tRNA(Arg) abundance. Other inactivating mutations were located in the anticodon loop, where they did not affect transcription and aminoacylation but probably altered interaction with the translation machinery. No lethal effects were observed when residues 16, 20 and 38 were individually mutated, despite the fact that they are involved in sequence-specific interactions with the aminoacyl-tRNA synthetase. However, the steady-state levels of the aminoacylated forms of U20A and U20G were decreased by a factor of 3.5-fold in vivo. This suggests that, unlike in the Escherichia coli tRNA(Arg):ArgRS system where residue 20 (A) is a major identity element, in yeast this position is of limited consequence.},
note = {1362-4962
Journal Article},
keywords = {Amino Acyl-tRNA Ligases/metabolism Arginine/genetics/metabolism Base Sequence Blotting, Arg/chemistry/*genetics/metabolism Saccharomyces cerevisiae/*genetics Support, ERIANI, Molecular Hydrogen-Ion Concentration Molecular Sequence Data Mutagenesis, Non-U.S. Gov't, Northern Cloning, Site-Directed Mutation Nucleic Acid Conformation RNA, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Here we report the construction of a yeast genetic screen designed to identify essential residues in tRNA(Arg). The system consists of a tRNA(Arg) knockout strain and a set of vectors designed to rescue and select for variants of tRNA(Arg). By plasmid shuffling we selected inactive tRNA mutants that were further analyzed by northern blotting. The mutational analysis focused on the tRNA D and anticodon loops that contact the aminoacyl-tRNA synthetase. The anticodon triplet was excluded from the analysis because of its role in decoding the Arg codons. Most of the inactivating mutations are residues involved in tertiary interactions. These mutations had dramatic effects on tRNA(Arg) abundance. Other inactivating mutations were located in the anticodon loop, where they did not affect transcription and aminoacylation but probably altered interaction with the translation machinery. No lethal effects were observed when residues 16, 20 and 38 were individually mutated, despite the fact that they are involved in sequence-specific interactions with the aminoacyl-tRNA synthetase. However, the steady-state levels of the aminoacylated forms of U20A and U20G were decreased by a factor of 3.5-fold in vivo. This suggests that, unlike in the Escherichia coli tRNA(Arg):ArgRS system where residue 20 (A) is a major identity element, in yeast this position is of limited consequence.