Recognition of tRNA^(Cys) by the E. coli cysteinyl-tRNA synthetase: in vivo and in vitro studies

Citation

Komatsoulis, George A. (1993) Recognition of tRNA^(Cys) by the E. coli cysteinyl-tRNA synthetase: in vivo and in vitro studies. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/c5zd-j733. https://resolver.caltech.edu/CaltechTHESIS:12062012-143754549

Abstract

A study of the recognition of tRNA^(Cys) by E. coli cysteinyl-tRNA synthetase using in vivo and in vitro methods was performed. All three anticodon nucleotides, the discriminator U73, and some element(s) within the tertiary domain (the D stem/loop, the TΨC stem/loop and extra loop) are important for recognition; the anticodon stem and acceptor stem appear to contain no essential elements. A T7 RNA polymerase transcribed tRNA^(Cys) is a 5.5-fold worse substrate than native tRNA^(Cys)(in terms of the selectivity constant, k_cat/K_m) mainly due to an increase in K_m. This may reflect recognition of modified nucleotides or subtle effects on the folding of the tRNA. The greatest loss of specificity caused by mutation of a single nucleotide occurs when the discriminator U73 is changed; k_cat/K_m declines 3 to 4 orders of magnitude depending on the substitution. Mutations in the wobble nucleotide of the anticodon also cause reductions in the selectivity constant of 3 orders of magnitude, while mutations in the other anticodon nucleotides caused lesser effects. Interestingly, a C35A mutation 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 factor(s) which maintain tRNA specificity. Future in vitro experiments will attempt to explain the in vivo discrimination between the glycine, phenylalanine, and cysteine tRNAs by the cysteinyl-tRNA synthetase. Finally, these results suggest that the notion that a small set of isoacceptor specific elements define tRNA identity (the socalled "second genetic code") is incorrect. A better model is based on competition between synthetases for tRNA substrates which contain differing amounts of partially overlapping identity determinants.

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