Mapping the protein interaction network for TFIIB-related factor Brf1 in the RNA polymerase III preinitiation complex.

TFIIB-related factor Brf1 is essential for RNA polymerase (Pol) III recruitment and open-promoter formation in transcription initiation. We site specifically incorporated a nonnatural amino acid cross-linker into Brf1 to map its protein interaction targets in the preinitiation complex (PIC). Our cross-linking analysis in the N-terminal domain of Brf1 indicated a ...
pattern of multiple protein interactions reminiscent of TFIIB in the Pol active-site cleft. In addition to the TFIIB-like protein interactions, the Brf1 cyclin repeat subdomain is in contact with the Pol III-specific C34 subunit. With site-directed hydroxyl radical probing, we further revealed the binding between Brf1 cyclin repeats and the highly conserved region connecting C34 winged-helix domains 2 and 3. In contrast to the N-terminal domain of Brf1, the C-terminal domain contains extensive binding sites for TBP and Bdp1 to hold together the TFIIIB complex on the promoter. Overall, the domain architecture of the PIC derived from our cross-linking data explains how individual structural subdomains of Brf1 integrate the protein network from the Pol III active center to the promoter for transcription initiation.
Mesh Terms:
Binding Sites, Blotting, Western, Mutation, Protein Binding, Protein Interaction Mapping, Protein Interaction Maps, Protein Structure, Tertiary, RNA Polymerase III, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, TATA-Box Binding Protein, Transcription Factor TFIIB, Transcription Factor TFIIIB, Transcription Initiation, Genetic
Mol. Cell. Biol.
Date: Feb. 01, 2014
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