Aminoacylation of tRNA gene transcripts is strongly affected by 3'-extended and dimeric substrate RNAs

Kinetic parameters of aminoacylation by E. coli phenylalanyl-tRNA synthetase vary for phage T5 tRNA(Phe) gene transcript from 0.950 to 2.545 microM for Km and from 550 to 400 min(-1) for kcat. To reveal the source of this variability for various RNA preparations, homogeneity of the transcripts has been examined. Presence of 3' extensions and dimer formation in transcript preparations reduced the catalytic efficiency kcat/Km several-fold. We have shown that the proportion of dimers and 3'-extended transcripts in tRNA preparations is sensitive to single-base substitutions in tRNA. While wild-type phage T5 tRNA(Phe) gene transcript contains about half of dimeric molecules, for some mutants this value increases up to 90% or drops to 0%. Phage T5 tRNA(Phe) gene with anticodon stem nucleotide substitutions used as a template in run-off transcription produces 5 times less 3'-extended molecules than the wild-type gene. In view of all these results kinetic parameters of aminoacylation reaction for many wild-type and mutant tRNA gene transcripts should be reevaluated.