A cryptic DNA binding domain at the COOH terminus of TFIIIB70 affects formation, stability, and function of preinitiation complexes.
TFIIIC-dependent assembly of yeast TFIIIB on class III genes unmasks a high avidity of TFIIIB for DNA. TFIIIB contains TATA-binding protein (TBP), TFIIIB90/B", and TFIIIB70/Brf1, which is homologous to TFIIB. Using limited proteolysis, we have found that the COOH terminus of TFIIIB70 (residues 510-596) forms a protease-resistant domain that binds DNA tightly as seen by Southwestern, DNase I footprinting, and gel shift assays. Consistent with a role for this DNA binding activity, preinitiation complexes were formed less efficiently with truncated TFIIIB70 lacking the COOH-terminal domain and displayed an increased sensitivity to heparin. B' (TFIIIB70 + TBP).TFIIIC.DNA complexes were also particularly unstable. In addition, TFIIIB.TFIIIC.DNA complexes containing truncated TFIIIB70 were impaired in promoting transcription initiation.
Huet
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J
Service de Biochimie et Génétique Moléculaire, Commissariat à l'Energie Atomique, Saclay, F91191 Gif sur Yvette Cedex, France.
Conesa
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C
Carles
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Sentenac
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017173:Caenorhabditis elegans; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006493:Heparin; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D016324:Sequence Tagged Sites; D016385:TATA Box; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019474:Transcription Factors, TFIII; D014158:Transcription, Genetic; D000098832:Transcription Factor TFIIIC
C441028:BRF1 protein, human; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC; D006493:Heparin; D004247:DNA