tRNA structure and ribosomal function. II. Interaction between anticodon helix and other tRNA mutations.

Using multiply mutated tRNA genes, we have studied unusual coding by tRNAs that have altered nucleotides (nt) 27-43, which normally form the top base-pair of the anticodon helix. In vivo, nt 27-43 mutations accelerate non-canonical C-A coding at the third (3') codon position 14-fold, similar to the 40-fold stimulation originally shown for first (5') codon position non-canonical G-U pairing. Thus the effects of nt 27-43 generalize to a second type of unusual coding. Nt 27-43 changes have a similar relative effect on tRNA level, aminoacylation, and ribosomal activity, despite concurrent changes of the 3' anticodon nucleotide which alter coding. However, under conditions of efficient aminoacylation, only a fraction of these (potential missense) anticodon changes can be recovered, suggesting toxicity. Available data support the idea that the effects of nt 27-43 are not particular to one codon. A previously isolated D-arm mutation (G24A) has a similar coding effect, enhancing both first position G-U wobble up to 130-fold, the third position C-A mispairing 40-fold. Anticodon helix mutations at 27-43 have little effect on 3' or 5' miscoding in the presence of the G24A D-arm mutation, and reciprocally, the D-arm's effects are much diminished in the presence of the anticodon helix mutations. Because these two tRNA loci alter both types of aberrant coding, and because they are highly interdependent, they may exploit a similar mechanism, dependent on a similar effect on tRNA conformation. We suggest a relatively non-specific decrease in the ribosomal rejection rates for tRNAs altered at anticodon helix nucleotides 27 and 43. Thus coding via non-canonical pairings at both 5' and 3' ends of the codon-anticodon helix has a measurable rate in vivo. However, we find that normal tRNA structure minimizes the efficiency of this aberrant translation. To put these same findings in another light, tRNAs bearing identical anticodons, if altered in structure elsewhere, may translate the genetic code differently.