Interactions of Brf1 peptides with the tetratricopeptide repeat-containing subunit of TFIIIC inhibit and promote preinitiation complex assembly.

Transcription factor IIIC (TFIIIC) is an essential multisubunit assembly factor for RNA polymerase (Pol) III transcription of 5S rRNA, tRNA, and other genes that have similar promoter architectures. Upon binding to the promoters of these genes (with TFIIIA in the case of 5S genes), TFIIIC directs the assembly of the heterotrimeric initiation factor TFIIIB to the DNA upstream of the transcription start site (reviewed in references 11 and 32). The subunits of TFIIIB are loosely associated in the absence of DNA and are assembled sequentially by TFIIIC, beginning with the TFIIB-related factor Brf1. Subsequent binding of the TATA-binding protein, TBP, is followed by the SANT domain-containing protein Bdp1 to form an exceptionally stable TFIIIB-DNA complex in which the DNA is kinetically trapped (5, 14, 15, 21). The recruitment of both the Brf1 and Bdp1 subunits of TFIIIB is directed by the tetratricopeptide repeat (TPR)-containing subunit of TFIIIC, Tfc4 (8, 23, 27). The interaction between Tfc4 and Brf1 represents a rate-limiting step in preinitiation complex assembly, as activating mutations in Tfc4 that increase Brf1 recruitment also facilitate TFIIIB complex assembly and Pol III transcription in vitro and increase the expression of a specific tRNA reporter gene in vivo (25, 27). The binding of Brf1 to Tfc4 is known to involve the N-terminal half of Tfc4 (Nt-TPR9), which contains two tandem TPR arrays (TPR1-5 and TPR6-9) separated by a 134-amino-acid intervening region (3, 28). Each TPR array can bind independently to Brf1 in vitro, and the significance of these interactions in the context of TFIIIC has been demonstrated genetically and biochemically (23, 25, 27). TPR motifs are found in functionally diverse proteins and have a general role as protein-protein interaction domains and in multisubunit complex assembly (7). Each TPR motif is composed of a degenerate sequence of 34 amino acids arranged as a pair of antiparallel α helices (designated A and B). The known structures of tandem TPRs, either free or in complex with ligands, reveal a similar superhelical architecture with a concave groove that provides or supports a site for ligand binding. Peptides as short as five amino acids bind in the superhelical groove in an extended conformation with moderate to low micromolar affinity (10, 31). This mode of binding together with the large number of TPR sequences in public databases suggests that the TPR protein family is likely to recognize many different peptides. At present, however, relatively few peptide ligands have been identified and the basis of their sequence-specific recognition has been determined in only a few cases (4, 10, 22, 31, 36). Guided by the available structures of TPR proteins, our genetic and biochemical studies of Tfc4 suggest that the superhelical groove of each TPR array provides binding sites for Brf1 (23, 25, 27, 28). Moreover, as there is little sequence similarity between the two TPR arrays in Tfc4 (apart from the residues that are important for the overall fold), each TPR array is predicted to interact with different sites in Brf1. Besides the binding sites in the TPR arrays, the N-terminal 128 amino acids of Tfc4 (Nt), which are essential for viability in Saccharomyces cerevisiae (8), are part of a high-affinity binding site for Brf1 (Nt-TPR5). Interestingly, the binding affinity of this site for Brf1, as well as the site present in TPR6-9, is significantly higher than that of a larger Tfc4 fragment (Nt-TPR9) that contains both of these sites (26). This has led to the suggestion that access of Brf1 to its binding sites in Tfc4 is limited by competing interactions within Nt-TPR9. This interpretation is supported by the behavior of a dominant mutation (the PCF1-1 mutation, H190Y) which maps to the convex surface of the first TPR array (25). The PCF1-1 mutation increases the affinity of the Nt-TPR9-Brf1 interaction but does not affect the interaction of Nt-TPR5 or TPR1-5 with Brf1. Thus, the mutation is thought to facilitate Brf1 binding by an indirect mechanism. Previous work has proposed that the PCF1-1 mutation relieves autoinhibition by stabilizing an alternative conformation of Tfc4 (25). Brf1 plays multiple roles in Pol III transcription. In addition to its participation in initiation complex assembly, which involves associations with Tfc4 and the other TFIIIB subunits, Brf1 functions in polymerase recruitment through interactions with Rpc34 and Rpc17 and is also important for promoter melting (1, 9, 18, 20, 35). These functions are mediated by multiple conserved domains in the TFIIB-related N terminus of Brf1 (Nt-Brf1) and in the Brf1-specific C-terminal half of the protein (Ct-Brf1). The two halves of Brf1 appear to be structurally and functionally independent, as TFIIIB-DNA complexes formed with Brf1 that is split into two fragments at residue 283 are transcriptionally active and are indistinguishable by footprinting from complexes formed with intact Brf1 (17). Moreover, a triple fusion protein that inserts the conserved core of TBP between the N- and C-proximal domains of Brf1 is able to replace Brf1 and TBP in transcription in vitro and can support viability of a BRF1 deletion strain (19). Interestingly, whereas each half of Brf1 is able to support TATA box-directed TFIIIB complex formation in the absence of TFIIIC, both halves of Brf1 are required for proper TFIIIC-directed complex assembly (17), indicating that the structural requirements on Brf1 are more stringent for TFIIIC-dependent reactions. Brf1 is not the only ligand for the TPRs of Tfc4; Bdp1 can also bind to Tfc4 fragments containing both TPR arrays (TPR1-9 or Nt-TPR9). A mutation (L469K) in TPR7 of Tfc4, which affects the recruitment of Brf1, also inhibits the direct binding of Bdp1 to Tfc4 and decreases the affinity of Bdp1 for the TBP-Brf1-TFIIIC-DNA complex, suggesting that Brf1 and Bdp1 share an overlapping binding site in Tfc4 (23). However, unlike Brf1, Bdp1 does not bind detectably to the individual TPR arrays or to TFIIIC-DNA. These data suggest that TFIIIC-dependent assembly of TFIIIB involves dynamic protein-protein interactions that likely include the repositioning of Brf1 to allow subsequent recruitment of Bdp1 (28). To better understand the molecular interactions between Brf1 and Tfc4 and the concerted binding of TFIIIB subunits to TFIIIC-DNA, we constructed Brf1 peptide libraries and used a yeast two-hybrid assay to identify peptides that bind to the high-affinity (Nt-TPR5) and autoinhibited (Nt-TPR9) binding sites of Tfc4. Two peptides identified against Nt-TPR5 were found to inhibit Brf1 recruitment by TFIIIC-DNA, and the corresponding regions in Brf1 were shown to be critical for this function. In contrast, a peptide identified against Nt-TPR9 increased Brf1-TFIIIC-DNA complex formation and affected complex mobility, consistent with a role in relieving autoinhibition within Tfc4. The results suggest a model for the dynamic interaction between Tfc4 and Brf1.