Investigation of promoter interference bytRNA genes in Saccharomyces cerevisiae.

Abstract

For my thesis project, I have investigated the effect of nearby tRNA genes on expression of several different RNA polymerase II genes in the yeast Saccharomyces cerevisiae. We have found that tRNA gene transcription represses transcription from adjacent RNA polymerase II promoters and have termed this phenomenon "promoter interference". Initial analysis of the mechanism of promoter interference was performed using an artificial plasmid construct in which a SUP4-ochre or SUP53-amber tRNA gene was placed immediately upstream of a chimeric RNA polymerase II gene in which a single GAL UAS and GAL1 promoter fragment activates transcription of the HIS3 gene protein coding region. When this construct is transformed into yeast, the SUP4 tRNA suppresses the ADE2-ochre mutation, but the level of expression from the GAL/HIS gene is not sufficient to complement a chromosomal HIS3 mutation. Experiments designed to probe the mechanism of promoter interference on this artificial plasmid construct suggest that an active RNA polymerase III transcription complex precludes the use of the RNA polymerase II promoter by indirect effects on DNA or nucleoprotein structure. The same artificial plasmid construct was used to isolate yeast mutants with altered promoter interference phenotypes which were able to grown on galactose media in the absence of histidine. In the only mutant examined extensively to date, a single gene defect has been shown to cause both the slow growth and HIS+ phenotypes. In addition, the rate of RNA polymerase III transcription of tRNA and 5S genes relative to RNA polymerase I transcription of 5.8S is significantly decreased in the mutant compared to its parental wild type. Further characterization of the mutants isolated should provide clues to the mechanism of promoter interference on this artificial plasmid construct as well as aid in the identification of factors involved in RNA polymerase III transcription. To address the physiological significance of the ability of a tRNA gene to repress transcription of an adjacent RNA polymerase II gene on an artificial plasmid construct, I asked whether or not promoter interference occurred between chromosomal tRNA genes and the closely associated repetitive elements sigma and delta. I found that tRNA genes do interfere with expression from the sigma and delta promoters, and probed the possible beneficial effects of this repression on yeast cell viability.