Transfer RNA structure and coding specificity. I. Evidence that a D-arm mutation reduces tRNA dissociation from the ribosome

Abstract

The mutation G to A24 in the D-arm of Escherichia coli tRNA(Trp) or its UAG suppressor derivative Su7 has two known phenotypes: (1) an altered or relaxed coding specificity at the codon third position; and (2) partial rescue of an anticodon loop mutation. In order to study the mechanism responsible for these effects we constructed, by in vitro mutagenesis, a series of tRNAs with alterations in the anticodon loop or at the third position of the anticodon. Evaluation of the effects of the A24 mutation on the in vivo ribosomal activity of these tRNAs leads us to conclude that the mutation reduces the rate at which the ribosome is able to reject tRNAs that are structurally defective or non-cognate. The apparent interaction of the D-arm mutation with the anticodon and anticodon loop is thus primarily kinetic, rather than through the structure of the tRNA. The Appendix describes the calculation of tRNA ribosomal activity from in vivo measurement of suppression efficiency.