Attempted expression of a human initiator tRNA gene in Saccharomyces cerevisiae

Abstract

In attempts to overproduce the wild type and, eventually, mutant human initiator methionine tRNAs for use in structure-function relationship studies, we have investigated the expression of the wild type human initiator tRNA gene in the yeast Saccharomyces cerevisiae, both in vitro and in vivo. We find that the yeast extract, while capable of accurately transcribing several yeast tRNA genes, does not transcribe the human initiator tRNA gene. In addition, when the human initiator tRNA gene is introduced into yeast as part of a 2 mu vector, no expression of the human tRNA gene was detected. A yeast alanine tRNA gene similarly introduced into yeast is expressed efficiently. The block in expression of the human tRNA gene is at the level of transcription and not processing. The yeast cell-free extract can accurately process precursors of the same human initiator tRNA made in a HeLa cell-free extract. Surprisingly, although the human tRNA gene has essentially the same intragenic control elements as the yeast initiator tRNA gene, the human tRNA gene competes extremely poorly for transcription factors in yeast extracts. In the course of screening a yeast DNA bank for initiator tRNA clones we have isolated and sequenced three yeast tRNA genes corresponding to glycine, alanine, and aspartic acid tRNAs. The sequence of glycine tRNA gene differs from the published tRNA sequence in having an additional nucleotide in the variable loop. The alanine tRNA gene codes for a new tRNA. All three genes are transcriptionally active in yeast extracts.