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Recognition of tRNA^(Cys) by Escherichia coli Cysteinyl-tRNA Synthetase

Komatsoulis, George A. and Abelson, John (1993) Recognition of tRNA^(Cys) by Escherichia coli Cysteinyl-tRNA Synthetase. Biochemistry, 32 (29). pp. 7435-7444. ISSN 0006-2960. https://resolver.caltech.edu/CaltechAUTHORS:20180207-085629031

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Abstract

A study of the recognition of tRNA^(CyS) by Escherichia coli cysteinyl-tRNA synthetase using in vivo and in vitro methods was performed. All three anticodon nucleotides, the discriminator nucleotide (73), and some elements within the tertiary domain (the D stem/loop, the TΨC stem/loop, and the variable loop) are important for recognition; the anticodon stem and acceptor stem appear to contain no essential elements. A T7 RNA polymerase transcript corresponding to tRNA^(Cys) is only a 5.5-fold worse substrate than native tRNA^(Cys) (in terms of the specificity constant, K_(cat)/K_m), mainly due to an increase in the value of K_m for the transcript. The greatest loss of specificity caused by mutation of a single nucleotide occurs when the discriminator U73 ischanged; k_(cat)/K_m declines 3 4or ders of magnitude depending on the substitution. Mutations in the wobble nucleotide of the anticodon also cause reductions in the specificity constant of 3 orders of magnitude, while mutations in the other anticodon nucleotides caused lesser effects. Interestingly, a C35A mutation (with the phenylalanine anticodon GAA) had no effect on aminoacylation by the cysteinyl-tRNA synthetase. Several amber suppressor tRNAs were constructed whose in vivo identity did not correlate with their in vitro specificity, indicating the need for both types of experiments to understand the factors which maintain tRNA specificity.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/bi00080a014DOIArticle
https://pubs.acs.org/doi/10.1021/bi00080a014PublisherArticle
Additional Information:© 1993 American Chemical Society. Published in print 1 July 1993. Supported by N.I.H. Grant No. GM32637-10 to J.N.A. and N.I.H. training Grant No. 5 T32 GM07616 to G.A.K. We thank D. Horowitz, C. O'Day, M. Saks, and J. Sampson for advice and/or critical examination of the manuscript.
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Funding AgencyGrant Number
NIHGM32637-10
NIH Predoctoral Fellowship5 T32 GM07616
Issue or Number:29
Record Number:CaltechAUTHORS:20180207-085629031
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180207-085629031
Official Citation:Recognition of tRNACys by Escherichia coli cysteinyl-tRNA synthetase George A. Komatsoulis and John Abelson Biochemistry 1993 32 (29), 7435-7444 DOI: 10.1021/bi00080a014
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:84707
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:07 Feb 2018 17:08
Last Modified:03 Oct 2019 19:21

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