The assembly of a transcription factor
(TF) IIIB-DNA complex by promoter-bound TFIIIC is initiated by its
tetratricopeptide repeat (TPR)-containing subunit, TFIIIC131, which
interacts directly with the TFIIB-related factor, TFIIIB70/Brf1.
Biochemical studies on several dominant activating mutations in TPRs 1-3
of TFIIIC131 have shown that these mutations increase transcription by
facilitating the recruitment of TFIIIB70 to TFIIIC.DNA. The activating
mutations do not appear to increase the affinity of mutant TFIIIC.DNA
complexes for TFIIIB70 but increase the number of complexes able to
recruit TFIIIB70. The data suggest a novel mechanism of activation in
which the TPR mutations facilitate a conformational change in TFIIIC
that is required for TFIIIB70 binding. To further explore this
mechanism, wild-type and mutant ( PCF1-1 ) fragments of TFIIIC131
were expressed in E. coli and purified to homogeneity. These
fragments exhibit a differential ability to function as inhibitors of
TFIIIB.DNA complex assembly that is consistent with the PCF1-1
phenotype. Circular dichroism spectroscopy of the mutant and wild-type
fragments reveals that the PCF1-1 mutation affects the structure
of the native protein, increasing its helical content. The results
provide direct support for the hypothesis that the mechanism of
activation by dominant PCF1 mutations involves a protein
conformational change in TFIIIC131.
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