The Brf1 and Bdp1 Subunits of Transcription Factor TFIIIB Bind to Overlapping Sites in the Tetratricopeptide Repeats of Tfc4

Abstract

The RNA polymerase III initiation factor TFIIIB is assembled onto DNA through interactions involving the Tfc4 subunit of the assembly factor TFIIIC and two subunits of TFIIIB, Brf1 and Bdp1. Tfc4 contains two arrays of tetratricopeptide repeats (TPRs), each of which provides a binding site for Brf1. Dominant mutations in the ligand binding channel of the first TPR array, TPRs1-5, and on the back side of this array, increase Brf1 binding by Tfc4. Here we examine the biological importance of the second TPR array, TPRs6 -9. Radical mutations at phylogenetically conserved residues in the ligand binding channel of TPRs6 -9 impair pol III reporter gene transcription. Biochemical studies on one such mutation, L469K in TPR7, revealed a defect in the recruitment of Brf1 into TFIIIB-TFIIIC-DNA complexes and diminished the direct interaction between Tfc4 and Brf1. Multicopy suppression analysis implicates TPR9 in Brf1 binding and TPRs7 and 8 in binding to more than one ligand. Indeed, the L469K mutation also decreased the binding affinity for Bdp1 incorporation into TFIIIB-TFIIIC-DNA complexes and inhibited binary interactions between Bdp1 and Tfc4. The Bdp1 binding domain in Tfc4 was mapped to TPRs1-9, a domain that contains both TPR arrays and thus overlaps two of the known binding sites for Brf1. The properties of the L469K mutation identify both Brf1 and Bdp1 as ligands for the second TPR array.The assembly of the initiation factor TFIIIB by TFIIIC is a limiting step in RNA polymerase III (pol III) 1 transcription. Although multiple subunits of both TFIIIB and TFIIIC interact (1-4) TFIIIB assembly is mediated initially by protein-protein interactions between the TFIIIC subunit, Tfc4, and the TFIIIB subunit, Brf1. Biochemical and genetic studies in yeast indicate that the recruitment of Brf1 by TFIIIC is a major limiting step in preinitiation complex assembly (5-7). Subsequent binding of the TBP and Bdp1 subunits of TFIIIB generates a series of structural changes in Tfc4, Brf1 and DNA that leads to progressively increased accessibility of Brf1 to DNA (8). The protein-protein interactions between TFIIIB subunits, together with DNA bending, generate a highly stable TFIIIB complex, a structure that surrounds and kinetically traps the DNA (9 -12).Tfc4 contains eleven copies of a ubiquitous protein-protein interaction motif known as a tetratricopeptide repeat (13,14).TPR motifs, as the name implies, are typically 34 amino acids in length and fold into two antiparallel ␣-helices (designated A and B, Ref. 15). Although no position within the motif is invariant, a pattern of small and large hydrophobic residues defines the loose TPR consensus and provides stacking interactions between adjacent repeats (15-17). Multiple adjacent repeats form a right-handed superhelix in which the inner channel (formed by the A-helices) provides a ligand binding interface (18 -20). The TPRs in Tfc4 are organized into two arrays in the N terminus, TPR1-5 and TPR6 -9 (with five and four repeats, respectively) that are separated by a region of mini...

Interactions of Brf1 Peptides with the Tetratricopeptide Repeat-Containing Subunit of TFIIIC Inhibit and Promote Preinitiation Complex Assembly

“…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.…”

“…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.…”

“…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).…”

“…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).…”

Motif clustering with implications for transcription factor interactions

“…Notably, RNA-polymerase III is connected to a completely different set of basal TFs, some of which are known to be important for RNA-polymerase III-dependent transcription (Liao et al, 2003).…”