Molecular genetic studies of PCF1-1: The yeast gene encoding the second largest subunit of TFIIIC

Abstract

Previous studies have shown that the PCF1-1 mutation of Saccharomyces cerevisiae suppresses the negative effect of a tRNA gene A box promoter mutation in vivo and increases the transcription of a variety of RNA polymerase (pol) III genes in vitro (Willis et al., 1989; 1992). This work reports the cloning and molecular genetic analysis of PCF1-1. The gene encodes an open reading frame of 1025 amino acids with a deduced size of 120 kDa. The PCF1-1 mutation causes an His{dollar}\to{dollar}Tyr amino acid substitution at position 190 in a novel protein structural motif, a tetratricopeptide repeat (TPR), in the protein. Tetrad and random spores analyses have shown that PCF1 is essential for cell viability.;PCF1p comigrates with the second largest subunit of TFIIIC which has been detected using antibodies provided by Parsons and Weil. DNA mobility shift and immunoprecipation experiments demonstrate that PCF1p is a subunit of the TFIIIC complex. Finally, the base sequence of PCF1 is identical to TFC4, the gene encoding the second largest subunit of TFIIIC.;In vitro transcription studies indicate that PCF1-1p facilitates a rate-limiting step in transcription, namely, the recruitment of TFIIIB to the template. This is suggested by and an elevated amount of the stoichiometrically limiting TFIIIB{dollar}\sb{lcub}70{rcub}{dollar} in these fractions. Thus, it is proposed that the PCF1-1 mutation modulates pol III trancription in vivo by increasing the rate of recruitment of TFIIIB to the template DNA (Rameau et al., 1994; Lopez-de-leon et al., 1992).