Site-specific mutagenesis on a human initiator methionine tRNA gene within a sequence conserved in all eukaryotic initiator tRNAs and studies of its effects on in vitro transcription.

We have used oligonucleotide-directed site-specific mutagenesis to generate a mutant human initiator tRNA gene in which the sequence GATCG corresponding to the universal GAUCG found in loop IV of eukaryotic cytoplasmic initiator tRNAs is changed to GTTCG. The mutant tRNA gene has been characterized by restriction mapping and by DNA sequencing. We show that this mutation has no effect on in vitro transcription of the tRNA gene in HeLa cell extracts. Transcripts derived from both the wild type (A54) and the mutant (T54) initiator tRNA genes are processed in vitro to produce mature tRNAs with the correct 5'-and 3'-termini. Fingerprint analysis of in vitro transcripts shows that the mutant RNA has the expected nucleotide change. Modified nucleotide composition analyses on the RNAs show that when A54 is changed to U54, the neighboring nucleotide U55 is modified quantitatively to psi 55 in the in vitro extracts; U54 itself is partially modified to ribo-T. Other modified bases identified in the in vitro transcripts include m1G, m2G, m7G, D, and m5C.