Transcription factor IIIC (TFIIIC) plays an important role in assembling the initiation factor TFIIIB on genes transcribed by RNA polymerase III (Pol III). In Saccharomyces cerevisiae, assembly of the TFIIIB complex by promoter-bound TFIIIC is thought to be initiated by its tetratricopeptide repeat (TPR)-containing subunit, TFIIIC131, which interacts directly with the TFIIB-related factor, TFIIIB70/Brf1. In this work, we have identified 10 dominant mutations in TFIIIC131 that increase Pol III gene transcription. All of these mutations are found within a discrete 53-amino-acid region of the protein encompassing TPR2. Biochemical studies of one of the mutations (PCF1-2) show that the increase in transcription is due to an increase in the recruitment of TFIIIB70 to TFIIC-DNA. The PCF1-2 mutation does not affect the affinity of TFIIIC for DNA, and the differential in both transcription and TFIIIB complex assembly is observed at saturating levels of TFIIIB70. This indicates that mutant and wild-type TFIIIC-DNA complexes have the same affinity for TFIIIB70 and suggests that the increased recruitment of this factor is achieved by a nonequilibrium binding mechanism. A novel mechanism of activation in which the TPR mutations facilitate a conformational change in TFIIIC that is required for TFIIIB70 binding is proposed. The implications of this model for the regulation of processes involving TPR proteins are discussed.