A Tetratricopeptide Repeat Mutation in Yeast Transcription Factor IIIC₁₃₁ (TFIIIC₁₃₁) Facilitates Recruitment of Tfiib-Related Factor TFIIIB₇₀

Abstract

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, TFIIIC 131 , which interacts directly with the TFIIB-related factor, TFIIIB 70 /Brf1. In this work, we have identified 10 dominant mutations in TFIIIC 131 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 TFIIIB 70 to TFIIIC-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 TFIIIB 70 . This indicates that mutant and wild-type TFIIIC-DNA complexes have the same affinity for TFIIIB 70 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 TFIIIB 70 binding is proposed. The implications of this model for the regulation of processes involving TPR proteins are discussed.The multisubunit transcription factor IIIC (TFIIIC) is an assembly factor that directs the binding of the general initiation factor, TFIIIB, upstream of the transcription start site of RNA polymerase III (Pol III) genes (21, 37). On tRNA gene templates, TFIIIC interacts with intragenic A and B block promoter elements. High-affinity binding of TFIIIC to DNA is mediated by the B block. This element is positioned at variable distances from the start site and, in at least one example, can function, albeit less efficiently, in an inverted orientation (5). These properties, together with the requirement for TFIIIC in transcription from chromatin templates (antirepression), provide a conceptual view of TFIIIC as an enhancer binding factor (5, 6). TFIIIC, through interactions with the A block, also plays a role in start site selection. Typically, the start site is located about 20 bp upstream of the A block. However, its position can be shifted within a 20-to 30-bp window by altering the placement of TFIIIB on the DNA (7,10,12,15). Redirection of TFIIIB involves its TATA-binding protein (TBP) subunit, in addition to TFIIIC, and can be achieved by inserting, repositioning, or mutating an upstream TATA element. The conformational flexibility required for this variable placement of TFIIIB has been suggested to reside in the TFIIIB-assembling subunit of TFIIIC, TFIIIC 131 (15).Specific transcription by Pol III can be achieved in the absence of TFIIIC on nucleosome-free templates which contain a suitable TATA box and an initiation site. Transcription of the Saccharomyces cerevisiae U6 gene, which contains the ...

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

“…Previous work has established that transcription of the sup9-e A19-supS1 reporter gene in vivo is sensitive to the level of Brf1 as well as to the Brf1 binding activity of Tfc4 (5)(6)(7)33). Thus, it was interesting to find that only two of the mutations (S541I and L542G in TPR9, Fig.…”

“…Purity (77-80%) was confirmed by SDS-PAGE analysis using direct staining with Coomassie Blue and comparison to known protein standards (23). The preparation of recombinant TBP, Brf1, and Bdp1, yeast polymerase III and conditions for assaying transcription using whole cell extracts or purified components have been previously reported (6,24). The details for the assembly of TFIIIC-dependent TFIIIB or Brf1 complexes, quantitation and analysis of complex formation are all provided in detail in an earlier publication (23).…”

“…Dominant gain of function mutations in Tfc4 map in and around TPR2 and increase the recruitment of Brf1 to TFIIIC-DNA (6). A structural model of TPRs1-3 showed that the conserved residues and sites of the gain of function mutations cluster into two potential binding sites: one that traverses the ligand binding channel and another that lies across the back side of the array (29).…”

“…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)(6)(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).…”

Isolation and Cloning of Four Subunits of a Fission Yeast TFIIIC Complex That Includes an Ortholog of the Human Regulatory Protein TFIIICβ

“…The data indicate that the TFIIIC complex that was isolated from S. pombe is active. DISCUSSION TFIIIC is a multisubunit transcription factor that has been well characterized in S. cerevisiae and human in vitro transcription systems that is required for the synthesis of tRNAs (8,10,46,54). We isolated from S. pombe the coding sequences as well as the proteins themselves, representing four subunits of TFIIIC.…”