Analysis of acceptor stem base pairing on tRNA(Trp) aminoacylation and function in vivo

The role of acceptor stem base pairs in determining the identity of Escherichia coli tRNA(Trp) was examined by complementation of an E. coli strain containing a temperature-sensitive tRNA(Trp) gene (trpTts) and by monitoring aminoacylation levels in vivo. All derivatives of tRNA(Trp) containing substitutions at the first 3 base pairs in the acceptor stem complemented the trpTts mutation at the nonpermissive temperature (42 degrees C). However, three acceptor stem derivatives (tRNA(Trp)/C1.G72, tRNA(Trp)/C2.G71, and tRNA(Trp)/A3.U70) required overexpression for growth at 42 degrees C. Northern analysis of these derivatives following acid/urea gel electrophoresis showed no defects in tRNA aminoacylation at the nonpermissive temperature. Instead, these tRNAs appear to be defective in translation. This was suggested by the weak opal suppressor activities of the corresponding tRNA(UCATrp) derivatives. These results demonstrate that the three terminal acceptor stem base pairs do not contribute to the identity of tRNA(Trp). Substitution of the C1.A72 base pair in a methionine initiator tRNA containing the tryptophan anticodon and discriminator base (tRNA(CCAfMet)/G73) with A1.U72, the base pair found in tRNA(Trp), or G1.C72 resulted in the conversion of these tRNAs into tryptophan-inserting elongator tRNAs in vivo. However, changes to U1.A72 or C1.G72 in tRNA(CCAfMet)/G73 resulted in misaminoacylation and/or defects in translation. Our data indicate that the A1.U72 base pair is a context-dependent, negative identity element of tRNA(Trp).