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10620.full - Proc Natl Acad Sci USA Biochemistry Vol 93 pp...

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Proc. Natl. Acad. Sci. USA Vol. 93, pp. 10620-10625, October 1996 Biochemistry High-resolution mapping of nucleoprotein complexes by site-specific protein-DNA photocrosslinking: Organization of the human TBP-TFIIA-TFIIB-DNA quaternary complex THIERRY LAGRANGE*, TAE-KYUNG KIM*, GEORGE ORPHANIDES*, YON W. EBRIGHTt, RICHARD H. EBRIGHTt, AND DANNY REINBERG*t *Howard Hughes Medical Institute, Department of Biochemistry, Division of Nucleic Acid Enzymology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854; and tDepartment of Chemistry and Waksman Institute, Rutgers University, New Brunswick, NJ 08855 Communicated by Keith R. Yamamoto, University of California, San Francisco, CA, July 11, 1996 (received for review May 7, 1996) ABSTRACT We have used a novel site-specific protein- DNA photocrosslinking procedure to define the positions of polypeptide chains relative to promoter DNA in binary, ter- nary, and quaternary complexes containing human TATA- binding protein, human or yeast transcription factor IIA (TFIIA), human transcription factor IIB (TFIIB), and pro- moter DNA. The results indicate that TFIIA and TFIIB make more extensive interactions with promoter DNA than previ- ously 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 assem- bly 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 con- tains 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 polypep- tides 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 N- terminal domain (11). However, the fully assembled transcription complex is too large for structure determination by NMR spec- troscopic and x-ray crystallographic methods. Therefore, infor- mation 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 biochem- ical methods (13-15).
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