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PMID: 8617245 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Transfer RNA-dependent cognate amino acid recognition by an aminoacyl-tRNA synthetase.

The EMBO journal ·Vol. 15 ·No. 8 ·1996-04-15 ·Pages 1983-91

Hong KW, Ibba M, Weygand-Durasevic I, Rogers MJ, Thomann HU, Söll D

Abstract

An investigation of the role of tRNA in the catalysis of aminoacylation of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) has revealed that the accuracy of specific interactions between GlnRS and tRNAGln determines amino acid affinity. Mutations in GlnRS at D235, which makes contacts with nucleotides in the acceptor stem of tRNAGln, and at R260 in the enzyme's active site were found to be independent during tRNA binding but interactive for aminoacylation. Characterization of mutants of GlnRS at position 235, showed amino acid recognition to be tRNA mediated. Aminoacylation of tRNA(CUA)Tyr [tyrT (UAG)] by GlnRS-D235H resulted in a 4-fold increase in the Km for the Gln, which was reduced to a 2-fold increase when A73 was replaced with G73. These and previous results suggest that specific interactions between GlnRS and tRNAGln ensure the accurate positioning of the 3' terminus. Disruption of these interactions can change the Km for Gln over a 30-fold range, indicating that the accuracy of aminoacylation is regulated by tRNA at the level of both substrate recognition and catalysis. The observed role of RNA as a cofactor in optimizing amino acid activation suggests that the tRNAGln-GlnRS complex may be partly analogous to ribonucleoprotein enzymes where protein-RNA interactions facilitate catalysis.

MeSH Terms
Adenosine Triphosphate/metabolism Amino Acid Sequence Base Sequence Binding Sites Escherichia coli/genetics,metabolism Glutamate-tRNA Ligase/chemistry,genetics,metabolism Glutamine/metabolism Kinetics Models, Molecular Molecular Sequence Data Mutagenesis, Site-Directed Point Mutation RNA, Bacterial/genetics,metabolism RNA, Transfer, Gln/genetics,metabolism Substrate Specificity Thermodynamics
Chemicals
RNA, Bacterial RNA, Transfer, Gln Glutamine Adenosine Triphosphate Glutamate-tRNA Ligase
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Hong K W
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
Ibba M
Weygand-Durasevic I
Rogers M J
Thomann H U
Söll D
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1996-04-15
Pages
1983-91
Language
English
Region
England
NLM ID
8208664
PMCID
PMC450117
Subset
IM
External Links
PubMed source PMC Full Text View journal details
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