The Brf1 and Bdp1 subunits of transcription factor TFIIIB bind to overlapping sites in the tetratricopeptide repeats of Tfc4.
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.
Liao
Yanling
Y
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Willis
Ian M
IM
Moir
Robyn D
RD
D001665:Binding Sites; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004258:DNA Polymerase III / Q000235:genetics; D017353:Gene Deletion; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013696:Temperature; D035361:Transcription Factor TFIIIB / Q000235:genetics / Q000378:metabolism*; D019474:Transcription Factors, TFIII / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D020798:Two-Hybrid System Techniques; D000098832:Transcription Factor TFIIIC
C090956:BRF1 protein, S cerevisiae; C474805:Bdp1 protein, S cerevisiae; D021122:Protein Subunits; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D035361:Transcription Factor TFIIIB; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC; D004247:DNA; D004258:DNA Polymerase III