Interactions of Brf1 peptides with the tetratricopeptide repeat-containing subunit of TFIIIC inhibit and promote preinitiation complex assembly.
The binding of Brf1 to the tetratricopeptide repeat (TPR)-containing transcription factor IIIC (TFIIIC) subunit (Tfc4) represents a rate-limiting step in the ordered assembly of the RNA polymerase III initiation factor TFIIIB. Tfc4 contains multiple binding sites for Brf1 within its amino terminus and adjacent TPR arrays, but the access of Brf1 to these sites is limited by autoinhibition. Moreover, the Brf1 binding sites in Tfc4 overlap with sites important for the subsequent recruitment of another TFIIIB subunit, Bdp1, implying that repositioning of Brf1 is required after its initial interaction with Tfc4. As a starting point for dissecting the steps in TFIIIC-directed assembly of TFIIIB, we conducted yeast two-hybrid screens of Brf1 peptide libraries 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 TPR6-9 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. The data are consistent with this peptide causing a conformational change in TFIIIC that overcomes Tfc4 autoinhibition of Brf1 binding and suggest a structural model for the Brf1-Tfc4 interaction.
Liao
Yanling
Y
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA.
Moir
Robyn D
RD
Willis
Ian M
IM
D000595:Amino Acid Sequence; D004271:DNA, Fungal / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D035381:TATA-Binding Protein Associated Factors; D035361:Transcription Factor TFIIIB / Q000737:chemistry / Q000378:metabolism*; D019474:Transcription Factors, TFIII / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D000098832:Transcription Factor TFIIIC
C090956:BRF1 protein, S cerevisiae; C441028:BRF1 protein, human; D004271:DNA, Fungal; D010455:Peptides; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D035361:Transcription Factor TFIIIB; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC