The role of non-coding DNA sequences in transcription and processing of a yeast tRNA

We have tested the hypothesis that conserved sequences in the intervening sequence (IVS) and 5'-flanking region of a yeast tRNALeu3 gene serve some function. Genes with deletions of 8, 10, 13 and 20 bp in the IVS are all active as templates in vitro. Yeast extracts produce mature tRNALeu3 from delta 8, delta 10 and delta 13 genes. Xenopus extracts do not detectably ligate the 5' and 3' half-molecules resulting from IVS excision. Neither extract is able to excise the IVS from delta 20 precursors. Genes with introns enlarged by 10, 21 or 30 bp of DNA produce mature tRNA. Insertion of 103 bp results in reduced levels of transcription, little if any end maturation, and no detectable mature product. A conserved 15 bp sequence is present at the 5'-end of the tRNA sequence. Replacement of yeast DNA up to position -22 leaves the tRNA gene transcriptionally active. With replacement extended to -2 the gene is active in Xenopus extracts but nearly inert in yeast extracts. We conclude that tRNA transcription in yeast is insensitive to IVS structure but can be positively influenced by 5'-flanking sequence.