Recessive mutations that activate transcription by RNA polymerase III in Saccharomyces cerevisiae

Abstract

Dominant and recessive mutations in the PCF1 gene, which encodes the 131 kDa subunit of transcription factor IIIC (TFIIIC{dollar}\sb{lcub}131{rcub}),{dollar} increase transcription by RNA polymerase III (pol III) in vivo and in vitro. These data suggest that TFIIIC{dollar}\sb{lcub}131{rcub}{dollar} has dual regulatory functions. Sequence analysis of two recessive alleles, pcf1-3, (R728K) and pcf1-59, (P906L) showed that the mutations lie towards the carboxy terminal end of the protein. In contrast, the dominant mutations characterized to date are located at the amino terminus. A structure-function analysis conducted at the site of the pcf1-3 mutation revealed that the mutant phenotype could be obtained by diverse amino acids at this position. This is consistent with a loss of function and a negative role for amino acid 728 in the wild type protein.;Pol III transcription was examined during the growth cycle in yeast. A comparison of transcription in exponential and postdiauxic (pre-stationary) phase cell extracts, prepared from mutant (pcf1-4, R728H) and wild type strains, showed that the activity of the postdiauxic extracts is reduced to similar low levels. Thus yeast, like higher eukaryotes, down regulate the production of pol III transcripts in response to reduced cell growth rate. The data also indicate that amino acid 728 in TFIIIC{dollar}\sb{lcub}131{rcub}{dollar} does not play a role in growth regulation of pol III transcription. In vitro assays showed that the 20 fold differential in transcription activity between pcf1-4 exponential and postdiauxic phase extracts could be accounted for by changes in TFIIIB{dollar}\sb{lcub}70{rcub}{dollar} (10 fold) and TFIIIC (2 fold). Western blotting experiments revealed a 3.7 {dollar}\pm{dollar} 0.5-fold difference in the amount of TFIIIB{dollar}\sb{lcub}70{rcub}{dollar} and no change in the level of TFIIIC{dollar}\sb{lcub}131{rcub}{dollar} between these extracts. Therefore, growth regulation is achieved by a decrease in the amount and/or activity of TFIIIB{dollar}\sb{lcub}70{rcub}{dollar} and TFIIIC.