We have used a novel site-specific protein-DNA photocrosslinking procedure to define the positions of polypeptide chains relative to promoter DNA in binary, ternary, and quaternary complexes containing human TATAbinding protein, human or yeast transcription factor IIA (TFIIA), human transcription factor IIB (TFIIB), and promoter DNA. The results indicate that TFIIA and TFIIB make more extensive interactions with promoter DNA than previously anticipated. TATA-binding protein, TFHA, and TFIIB surround promoter DNA for two turns of DNA helix and thus may form a "cylindrical clamp" effectively topologically linked to promoter DNA. Our results have implications for the energetics, DNA-sequence-specificity, and pathway of assembly of eukaryotic transcription complexes.Transcription initiation at a eukaryotic protein-encoding gene requires assembly on promoter DNA of a transcription complex consisting of RNA polymerase II and six general transcription factors: TFIIA, TFIIB, TFIID [or TATA-binding protein (TBP)], TFIIE, TFIIF, and TFIIH (1). The smallest transcription complex generally competent for basal and activated transcription contains at least 35 distinct polypeptides and has a molecular mass in excess of 2 MDa (1). Understanding transcription initiation and transcription activation will require elucidation of the structures and the arrangement of these polypeptides relative to each other and to promoter DNA. Recently, structures have been determined for several polypeptides and polypeptide domains within the transcription complex: i.e., TBP core domain (TBPc), alone and in the TBPc-DNA binary complex (2-6); TFIIA in the TBPc-TFIIA-DNA ternary complex (7,8); TFIIB core domain (TFIIBc), alone and in the TBPc-TFIIBc-DNA ternary complex (9, 10); and TFIIB Nterminal domain (11). However, the fully assembled transcription complex is too large for structure determination by NMR spectroscopic and x-ray crystallographic methods. Therefore, information regarding the arrangement of polypeptides relative to each other and to promoter DNA within the fully assembled transcription complex must come from imaging (12) or biochemical methods (13-15).
EXPERIMENTAL STRATEGYIn this work, we have used a novel site-specific protein-DNA photocrosslinking procedure to determine the positions of individual polypeptides relative to promoter DNA within the complex. By "position," we refer to the translational position relative to the transcription start site, the rotational orientation relative to the DNA helix axis, and the groove orientation relative to the DNA major and minor grooves. Our procedure has four steps ( Fig. 1): (i) we use chemical (16-18) and enzymatic (19) reactions to prepare a promoter DNA fragment containing a photoactivatible crosslinking agent and an adjacent radiolabel incorporated at a single defined DNA phosphate; (ii) we form a protein-DNA complex using the site-specifically derivatized promoter DNA fragment, and we UV-irradiate the complex, initiating covalent crosslinking with polypeptide chains in dir...