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PMID: 1396597 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Recognition of bases in Escherichia coli tRNA(Gln) by glutaminyl-tRNA synthetase: a complete identity set.

The EMBO journal ·Vol. 11 ·No. 11 ·1992-11-00 ·Pages 4159-65

Hayase Y, Jahn M, Rogers MJ, Sylvers LA, Koizumi M, Inoue H, Ohtsuka E, Söll D

Abstract

The fidelity of protein biosynthesis rests largely on the correct aminoacylation of transfer RNAs by their cognate aminoacyl-tRNA synthetases. Previous studies have demonstrated that the interaction of Escherichia coli tRNA(Gln) with glutaminyl-tRNA synthetase (GlnRS) provides an excellent system for studying the basis of this highly specific recognition process. Correct aminoacylation depends on the set of nucleotides (identity elements) in tRNA(Gln) responsible for correct interaction with GlnRS. Specific contacts between tRNA(Gln) and GlnRS include the 2-amino group of guanosines. Therefore, we made a set of tRNA(Gln) variants in which specific guanosines were replaced by inosine using recombinant RNA technology. This resulted in a set of tRNAs that varied by single deletions of the amino group from guanine residues, thus allowing us to test the functional importance of these contacts. In addition, a number of mutants were made by transcription of mutated tRNA genes with base changes at position 10, 16 or 25. In vitro aminoacylation of these mutants showed decreases in the specificity constant (kcat/KM) of up to 300-fold, with kcat being the parameter most affected. These experiments reveal G10 as a new element of glutamine identity. In addition, the interaction of G2, G3 and G10 with GlnRS via the 2-amino group is significant for tRNA discrimination. Based on these results, and on earlier data, we propose a complete set of bases as identity elements for tRNA(Gln).

MeSH Terms
Amino Acid Sequence Amino Acyl-tRNA Synthetases/metabolism Base Sequence Escherichia coli/genetics,metabolism Hydrogen Bonding Kinetics Models, Molecular Molecular Sequence Data Mutagenesis Nucleic Acid Conformation Protein Conformation RNA, Transfer, Gln/genetics,metabolism Substrate Specificity Transcription, Genetic
Chemicals
RNA, Transfer, Gln Amino Acyl-tRNA Synthetases glutaminyl-tRNA synthetase
Authors & Affiliations
8 authors, click to expand affiliations / ORCID
Hayase Y
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
Jahn M
Rogers M J
Sylvers L A
Koizumi M
Inoue H
Ohtsuka E
Söll D
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1992-11-00
Pages
4159-65
Language
English
Region
England
NLM ID
8208664
PMCID
PMC556926
Subset
IM
Databases
GENBANK
X66364, X66365, X67872, X67873, X68629, X68630, Z14319, Z14320, Z14954, Z14955
External Links
PubMed source PMC Full Text View journal details
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