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

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