ABSTRACT
The human RNA polymerase II transcription factor B-TFIID consists of TATA-binding protein (TBP) and the TBP-associated factor (TAF) TAF(II)170 and can rapidly redistribute over promoter DNA. Here we report the identification of human TBP-binding regions in human TAF(II)170. We have defined the TBP interaction domain of TAF(II)170 within three amino-terminal regions: residues 2 to 137, 290 to 381, and 380 to 460. Each region contains a pair of Huntington-elongation-A subunit-Tor repeats and exhibits species-specific interactions with TBP family members. Remarkably, the altered-specificity TBP mutant (TBP(AS)) containing a triple mutation in the concave surface is defective for binding the TAF(II)170 amino-terminal region of residues 1 to 504. Furthermore, within this region the TAF(II)170 residues 290 to 381 can inhibit the interaction between Drosophila TAF(II)230 (residues 2 to 81) and TBP through competition for the concave surface of TBP. Biochemical analyses of TBP binding to the TATA box indicated that TAF(II)170 region 290-381 inhibits TBP-DNA complex formation. Importantly, the TBP(AS) mutant is less sensitive to TAF(II)170 inhibition. Collectively, our results support a mechanism in which TAF(II)170 induces high-mobility DNA binding by TBP through reversible interactions with its concave DNA binding surface.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , TATA-Binding Protein Associated Factors , Transcription Factor TFIID , Transcription Factors, TFII/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Drosophila , Glutathione Transferase/metabolism , Humans , Molecular Sequence Data , Plasmids/metabolism , Precipitin Tests , Promoter Regions, Genetic , Protein Binding , Protein Structure, Tertiary , Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , TATA-Box Binding Protein , Transcription, Genetic , Two-Hybrid System TechniquesABSTRACT
The Cockayne syndrome B protein (CSB) is required for coupling DNA excision repair to transcription in a process known as transcription-coupled repair (TCR). Cockayne syndrome patients show UV sensitivity and severe neurodevelopmental abnormalities. CSB is a DNA-dependent ATPase of the SWI2/SNF2 family. SWI2/SNF2-like proteins are implicated in chromatin remodeling during transcription. Since chromatin structure also affects DNA repair efficiency, chromatin remodeling activities within repair are expected. Here we used purified recombinant CSB protein to investigate whether it can remodel chromatin in vitro. We show that binding of CSB to DNA results in an alteration of the DNA double-helix conformation. In addition, we find that CSB is able to remodel chromatin structure at the expense of ATP hydrolysis. Specifically, CSB can alter DNase I accessibility to reconstituted mononucleosome cores and disarrange an array of nucleosomes regularly spaced on plasmid DNA. In addition, we show that CSB interacts not only with double-stranded DNA but also directly with core histones. Finally, intact histone tails play an important role in CSB remodeling. CSB is the first repair protein found to play a direct role in modulating nucleosome structure. The relevance of this finding to the interplay between transcription and repair is discussed.
Subject(s)
Adenosine Triphosphate/metabolism , Chromatin/metabolism , DNA Helicases/metabolism , DNA Repair , Nuclear Proteins , Nucleic Acid Conformation , Transcription, Genetic , Animals , Cell Extracts , Chromatin/chemistry , Chromatin/genetics , Cockayne Syndrome/genetics , DNA Helicases/genetics , DNA Repair Enzymes , DNA, Superhelical/chemistry , DNA, Superhelical/genetics , DNA, Superhelical/metabolism , DNA-Binding Proteins/metabolism , Deoxyribonuclease I/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/embryology , Gene Expression Regulation , HeLa Cells , Histones/chemistry , Histones/metabolism , Humans , Models, Genetic , Mutation , Nucleosomes/chemistry , Nucleosomes/genetics , Nucleosomes/metabolism , Plasmids/chemistry , Plasmids/genetics , Plasmids/metabolism , Poly-ADP-Ribose Binding Proteins , Recombinant Fusion Proteins , Transcription Factors/metabolism , Trypsin/metabolismABSTRACT
The TATA-binding protein (TBP) plays a central role in eukaryotic transcription and forms protein complexes with TBP-associated factors (TAFs). The genes encoding TAF(II) proteins frequently map to chromosomal regions altered in human neoplasias. TAF(II)170 of B-TFIID is a member of the SF2 superfamily of putative helicases. Members of this superfamily have also been implicated in several human genetic disorders. In this study we have isolated human genomic clones encoding TAF(II)170 and we show that the gene contains 37 introns. Ribonuclease-protection experiments revealed that TAF(II)170 has multiple transcription start sites, consistent with the observation that the promoter lacks a canonical TATA box and initiator element. Deletion analysis of the promoter region showed that a fragment of 264 bp is sufficient to direct transcription. In addition, we determined the chromosomal localization by two independent methods which mapped the gene to human chromosome 10q22-q23 between the markers D10S185 and WI-1183. The region surrounding these markers has been implicated in several human disorders.
