Studies of autoinhibition and TPR motifs in the assembly of a transcription pre-initiation complex
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The interaction between the tetratricopeptide repeat (TPR)-containing TFIIIC subunit, known as Tfc4 in yeast, and the TFIIB-related factor Brf1 represents a rate limiting step in the RNA polymerase III pre-initiation complex assembly. Tfc4 contains multiple binding sites for Brf1 within its amino terminus and adjacent two TPR arrays, yet the access of Brf1 to these sites is limited by autoinhibition. Previous studies on the first TPR array, TPRs1-5, have suggested that the superhelical groove of this array binds to Brf1 directly, whereas the surface on the backside of the groove is involved in an intramolecular interaction that is related to autoinhibition.;This thesis has demonstrated the biological importance of the second TPR array (TPRs6-9) in binding to Brf1. Moreover, the data has indicated that the Brf1 binding sites in Tfc4 overlap with the sites that interact with the SANT-domain containing TFIIIB subunit, Bdp1, implying that repositioning of Brf1 is required after its initial interaction with Tfc4.;To further dissect the steps in TFIIIC-directed assembly of TFIIIB, yeast two-hybrid screens of Brf1 peptide libraries were carried out against different TPR-containing Tfc4 fragments. Short, biochemically active peptides were identified in three distinct regions of Brf1. Two peptides defined conserved but distal regions of Brf1 that participate in stable binding of Brf1 to TFIIIC-DNA. Remarkably, a third peptide that binds specifically to TPRs6-9 of Tfc4 was found to promote the formation of both TFIIIC-DNA and Brf1-TFIIIC-DNA complexes and to reduce the mobility of these complexes in native gels, suggestive of a conformational change in TFIIIC that overcomes Tfc4 autoinhibition of Brf1 binding. In addition, some of the sites of interaction between Brf1 and Tfc4 that were defined by Brf1 peptides are also likely to be involved in steps that occur subsequent to TFIIIB assembly. All together, these data support the dynamic nature of the Brf1-Tfc4 interaction and are consistent with initial binding by Brf1 and its subsequent repositioning to allow the assembly of other TFIIIB subunits and the recruitment of pol III.
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