MUTATIONAL ANALYSIS OF THE TRANSCRIPTION START SITE OF THE YEAST TRNA(3)(LEU) GENE

In addition to the well-known internal promoter elements of tRNA genes , 5' flanking sequences can also influence the efficiency of transcrip tion by Saccharomyces cerevisiae extracts in vitro. A consensus sequen ce of yeast tRNA genes in the vicinity of the transcriptional start si te can be derived. To determine whether the activity of this region ca n be attributed to particular sequence features we studied in vitro mu tants of the start site region. We found that the start site can be sh ifted, but only to a limited extent, by moving the conserved sequence element. We found that both a pyrimidine-purine motif (with transcript ion initiating at the purine) and a small T:A base pair block upstream are important for efficient transcription in vitro. Thus the sequence surrounding the start site of transcription of the yeast tRNA(3)(Leu) gene does play a role in determining transcription efficiency and fix ing the precise site of initiation by RNA polymerase III.