Three regions of a yeast tRNALeu3 gene promote RNA polymerase III transcription

Abstract

A Saccharomyces cerevisiae tRNALeu3 gene has been dissected to identify sequences essential for recognition by the yeast RNA polymerase III transcription apparatus. Three putative promoter regions have been identified, one each in the 3'- and 5'-halves of the tRNA coding sequence, the A- and B-blocks, respectively, and one in the 5'-flanking region. DNA fragments derived from the intact gene and bearing the 5'-flanking region, the 5'-flanking region plus the A-block, the A- and B-blocks without the 5'-flanking region, the 5'-flanking region with the B-block, and the B-block only have been subcloned. Plasmids carrying these fragments were used as templates in a homologous in vitro transcription assay to determine the contributions of the various sequences to the template activity of the gene. No template-dependent transcription was seen when fragments with only the 5'-flanking region or B-block were tested. A very weak template-dependent transcript was observed from clones bearing the A- and B-block regions. Transcription of the fragment bearing the 5'-flanking sequence and A-block was considerably more efficient but reduced relative to the intact gene. The clone including the 5'-flanking region and 3'-half of the gene is transcribed by the yeast extract with an efficiency approaching that of the intact gene. Partial deletions were constructed in which the highly conserved 5'-flanking pentadecanucleotide sequence was replaced by vector DNA. Replacement of the sequence between positions -12 and -2 (relative to the tRNA coding sequence) decreased transcription efficiency 10-fold even though the A- and B-blocks were left intact. We conclude that this 5'-flanking region, in conjunction with either the A- or B-block sequence is sufficient to constitute a promoter for the yeast RNA polymerase III transcription apparatus.