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PMID: 10744027 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

In vivo selection of lethal mutations reveals two functional domains in arginyl-tRNA synthetase.

RNA (New York, N.Y.) ·Vol. 6 ·No. 3 ·2000-03-00 ·Pages 434-48

Geslain R, Martin F, Delagoutte B, Cavarelli J, Gangloff J, Eriani G

Abstract

Using random mutagenesis and a genetic screening in yeast, we isolated 26 mutations that inactivate Saccharomyces cerevisiae arginyl-tRNA synthetase (ArgRS). The mutations were identified and the kinetic parameters of the corresponding proteins were tested after purification of the expression products in Escherichia coli. The effects were interpreted in the light of the crystal structure of ArgRS. Eighteen functional residues were found around the arginine-binding pocket and eight others in the carboxy-terminal domain of the enzyme. Mutations of these residues all act by strongly impairing the rates of tRNA charging and arginine activation. Thus, ArgRS and tRNA(Arg) can be considered as a kind of ribonucleoprotein, where the tRNA, before being charged, is acting as a cofactor that activates the enzyme. Furthermore, by using different tRNA(Arg) isoacceptors and heterologous tRNA(Asp), we highlighted the crucial role of several residues of the carboxy-terminal domain in tRNA recognition and discrimination.

MeSH Terms
Arginine-tRNA Ligase/chemistry,genetics Cloning, Molecular Fungal Proteins/biosynthesis,genetics Gene Expression Regulation, Fungal Genes, Fungal/genetics Genes, Lethal/genetics Kinetics Models, Molecular Mutation/genetics Peptide Fragments/chemistry,genetics Saccharomyces cerevisiae/enzymology,genetics
Chemicals
Fungal Proteins Peptide Fragments Arginine-tRNA Ligase
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Geslain R
Unité Propre de Recherche 9002 Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Strasbourg, France.
Martin F
Delagoutte B
Cavarelli J
Gangloff J
Eriani G
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Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1355-8382
DOI
10.1017/s1355838200992331
Published
2000-03-00
Pages
434-48
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC1369925
Subset
IM
External Links
PubMed source DOI: 10.1017/s1355838200992331 PMC Full Text View journal details
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