A mutation in the second largest subunit of TFIIIC increases a rate-limiting step in transcription by RNA polymerase III.

In previous studies, we have shown that the PCF1-1 mutation of Saccharomyces cerevisiae suppresses the negative effect of a tRNA gene A block promoter mutation in vivo and increases the transcription of a variety of RNA polymerase III genes in vitro. Here, we report that PCF1 encodes the second largest subunit of transcription factor IIIC (TFIIIC) and that the PCF1-1 mutation causes an amino acid substitution in a novel protein structural motif, a tetratricopeptide repeat, in this subunit. In agreement with the nature of the mutation, in vitro transcription studies with crude extracts indicate that PCF1-1 facilitates the rate-limiting step in transcription, namely, the recruitment of TFIIIB to the template. Additionally, biochemical fractionation of wild-type and mutant cell extracts shows that PCF1-1 increases the amount of the 70-kDa TFIIIB subunit detectable by Western (immunoblot) analysis in purified TFIIIB fractions and the transcription activity of a TFIIIB" fraction containing the 90-kDa subunit of this factor. We suggest that the effect of PCF1-1 on TFIIIB activity in vitro is a consequence of its increased rate of recruitment in vivo.

Recessive mutations in the second largest subunit of TFIIIC suggest a new step in RNA polymerase III transcription.

An analysis of mutant S. cerevisiae strains selected for their ability to increase transcription by RNA polymerase (pol) III has identified 14 isolates in which this phenotype is recessive. Genetic linkage and complementation studies suggest that all 14 isolates contain recessive alleles of PCF1. PCF1 encodes the 131-kDa subunit of transcription factor IIIC (TFIIIC131) and was identified previously by dominant mutations that also increased transcription by pol III. The recessive mutation, pcf1-3, results in a conservative substitution (R728-->K) towards the carboxyl-terminus of the protein. This position is distinct from the site of the dominant mutation PCF1-1 (H190-->Y), which maps to a tetratricopeptide repeat (TPR). Site-directed mutagenesis at amino acid 728 generated one allele, pcf1-4, with a stronger phenotype than pcf1-3. Extracts from pcf1-3 and pcf1-4 strains increase pol III transcription two- to threefold and ninefold, respectively, over wild-type under conditions that permit either single or multiple rounds of initiation. The entire effect of these mutations in vitro can be accounted for by an increase in the amount of transcriptionally active TFIIIB. In contrast, PCF1-1 primarily affects the rate of preinitiation complex assembly. The genetic, molecular, and biochemical data suggest that amino acid 728 in TFIIIC131 constitutes part of a structural domain in this protein that affects TFIIIB activity by influencing a previously undefined step in transcription. This step is suggested to occur after the recruitment of TFIIIB to DNA.

In vitro evidence for growth regulation of tRNA gene transcription in yeast. A role for transcription factor (TF) IIIB70 and TFIIIC.

We report in vitro studies showing that tRNA gene transcription in yeast is down-regulated during the transition from logarithmic to stationary phase growth. Transcription in a postdiauxic (early stationary) phase extract of a wild-type strain decreased 3-fold relative to a log phase extract. This growth stage-related difference in transcription was amplified to 20-fold in extracts of a strain containing a mutation (pcf1-4) in the 131-kDa subunit of TFIIIC. The reduction in transcription activity in both wild-type and mutant postdiauxic phase extracts was correlated with a decrease in the amount of TFIIIB70, the limiting factor in these extracts. However, the 3.7 +/- 0.5-fold decrease in amount of TFIIIB70 in mutant extracts does not, by itself, account for the 20-fold decrease in transcription. Accordingly, transcription in the mutant postdiauxic phase extract could be reconstituted to a level equal to the mutant log phase extract by the addition of two components, TFIIIB70 and TFIIIC. Addition of TFIIIB70 increased transcription 10-fold, while a 2-fold effect of TFIIIC was seen at saturating levels of TFIIIB70. The data suggest that both TFIIIB70 and TFIIIC play a role in coordinating the level of polymerase III transcription with cell growth rate.