title: Nonradioactive, ultrasensitive site-specific protein-protein photocrosslinking: interactions of alpha-helix 2 of TATA-binding protein with general transcription factor TFIIA and transcriptional repressor NC2.
abstract: We have developed an approach that enables nonradioactive, ultrasensitive (attamole sensitivity) site-specific protein-protein photocrosslinking, and we have applied the approach to the analysis of interactions of alpha-helix 2 (H2) of human TATA-element binding protein (TBP) with general transcription factor TFIIA and transcriptional repressor NC2. We have found that TBP H2 can be crosslinked to TFIIA in the TFIIA-TBP-DNA complex and in higher order transcription-initiation complexes, and we have mapped the crosslink to the 'connector' region of the TFIIA alpha/beta subunit (TFIIAalpha/beta). We further have found that TBP H2 can be crosslinked to NC2 in the NC2-TBP-DNA complex, and we have mapped the crosslink to the C-terminal 'tail' of the NC2 alpha-subunit (NC2alpha). Interactions of TBP H2 with the TFIIAalpha/beta connector and the NC2alpha C-terminal tail were not observed in crystal structures of TFIIA-TBP-DNA and NC2-TBP-DNA complexes, since relevant segments of TFIIA and NC2 were not present in truncated TFIIA and NC2 derivatives used for crystallization. We propose that interactions of TBP H2 with the TFIIAalpha/beta connector and the NC2alpha C-terminal tail provide an explanation for genetic results suggesting importance of TBP H2 in TBP-TFIIA interactions and TBP-NC2 interactions, and provide an explanation-steric exclusion-for competition between TFIIA and NC2.
authors.affiliation: Howard Hughes Medical Institute, Waksman Institute, and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway NJ 08854, USA.
authors.forename: Younggyu
authors.initials: Y
authors.lastname: Kim
authors.forename: Yon W
authors.initials: YW
authors.lastname: Ebright
authors.forename: Adam R
authors.initials: AR
authors.lastname: Goodman
authors.forename: Danny
authors.initials: D
authors.lastname: Reinberg
authors.forename: Richard H
authors.initials: RH
authors.lastname: Ebright
mesh_terms: D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000528:radiation effects; D004247:DNA / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D012319:RNA Polymerase II / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D035181:TATA-Box Binding Protein / Q000737:chemistry* / Q000378:metabolism; D035561:Transcription Factor TFIIA / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D019473:Transcription Factors, TFII / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D007202:Indicators and Reagents; D010750:Phosphoproteins; D012097:Repressor Proteins; D035181:TATA-Box Binding Protein; C509817:TBP protein, human; D035561:Transcription Factor TFIIA; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C075995:down-regulator of transcription 1; D004247:DNA; D012319:RNA Polymerase II