Because T7 RNA polymerase has a strong preference for particular sequences to initiate transcription, some RNAs having pyrimidine-rich sequences at their 5'-end (yeast tRNA(Tyr), for example) are hardly transcribed by this enzyme. To circumvent this inconvenience, we have developed an efficient method for in vitro preparation of such tRNAs. The RNA of interest is first transcribed as a precursor form that has purine-rich extra sequences at its 5'-end, then processed with RNase P to generate the objective tRNAs. By using this protocol, we were able to prepare easily and efficiently yeast tRNA(Tyr) transcript and its mutants harboring base substitutions within the anticodon loop and/or acceptor stem regions. Aminoacylation analyses of these tRNA transcripts with yeast tyrosyl-tRNA synthetase revealed that the replacement of G34 by C34 (mutation to amber suppressor) severely impaired the aminoacylation, whereas the replacement of the U4:G69 wobble base-pair in the acceptor stem region by C4:G69 normal Watson-Crick type base-pair improved it.