Activation of RNA polymerase III transcription by TFIIIC(131) and TFIIIB(70) in the yeast Saccharomyces cerevisiae

Abstract

In the yeast Saccharomyces cerevisiae transcription by RNA polymerase III requires two multisubunit factors, TFIIIB and TFIIIC, one gene-specific factor TFIIIA and another factor, TFIIIE, whose composition is unknown. To study the structure, function and regulation of these factors, a collection of transcriptionally altered dominant mutant strains was generated in which the deleterious effects of an A-block promoter mutation were reversed. This dissertation presents a molecular and biochemical characterization of two genes, PCF1-2 and PCF4, isolated from strains in this collection.;The PCF1-2 mutation is a cytosine to thymine transition 500 nucleotides downstream from the translation start site and results in a threonine to isoleucine change at amino acid 167. PCF1-2 whole cell extracts show a several increase in Pol III transcription. The transcription of all class III genes analyzed to date is increased two to eight fold in mutant cell extracts. Single round transcription assays showed that the PCF1-2 mutation increases the number of active transcription complexes. Consistent with this result, extract mixing experiments demonstrated that the PCF1-2 mutation increases the activity of a stoichiometrically-required, limiting initiation factor. Since TFIIIB is the limiting factor in whole-cell extracts, these results suggest that PCF1-2 directly or indirectly increases the activity of this factor. This was confirmed in transcription reconstitution assays using TFIIIB, TFIIIC and Pol III fractions purified from wild-type and PCF1-2 cell extracts.;The PCF4 gene was isolated from a genomic bank of the mutant strain #18-10. Molecular genetic experiments showed that the cloned gene is wild type and that it was isolated by a gene dosage effect. Additional genetic and biochemical studies performed with a PCF4 over-expressor strain demonstrated that the gene product is limiting for transcription in vivo and in vitro. The amino-terminal half of the PCF4 gene product has a high degree of homology to the RNA polymerase II transcription factor TFIIB. This observation, together with the size and the biochemical properties of PCF4, suggest that it encodes the 70 kDa subunit of TFIIIB. The gene product has all the properties expected for a polymerase-specificity factor.