A MUTATION IN THE 2ND 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 Sacchar omyces cerevisiae suppresses the negative effect of a tRNA gene A bloc k promoter mutation in vivo and increases the transcription of a varie ty of RNA polymerase III genes in vitro. Here, we report that PCF1 enc odes the second largest subunit of transcription factor IIIC (TFIIIC) and that the PCF1-1 mutation causes an amino acid substitution in a no vel protein structural motif, a tetratricopeptide repeat, in this subu nit. In agreement with the nature of the mutation, in vitro transcript ion studies with crude extracts indicate that PCF1-1 facilitates the r ate-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 tha t the effect of PCF1-1 on TFIIIB activity in vitro is a consequence of its increased rate of recruitment in vivo.