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 (trpT_ts) 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 trpT_ts mutation at the nonpermissive temperature (42 °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 °C. Northern analysis of these derivatives following acid/urea gel electrophoresis showed no defects in tRNA aminoacylation at the nonpermissive temperature. Instead, these tRN As appear to be defective in translation. This was suggested by the weak opal suppressor activities of the corresponding tRNA_UCA^Trp 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_CCA^fMet/G₇₃) with A₁·U₇₂, the base pair found in tRNA^Trp, or G₁·C₇₂ resulted in the conversion of these tRN As into tryptophan-inserting elongator tRNAs in vivo. However, changes to U₁·A₇₂ or C₁·G₇₂ in tRNA_CCA^fMet/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.