High-resolution protein-DNA contacts for the yeast RNA polymerase II general transcription machinery.
We used site-specific protein-DNA photo-cross-linking to define contact points between Saccharomyces cerevisiae RNA polymerase II (RNAP II) and the general transcription factors TBP, TFIIB, and TFIIF on promoter DNA. We present three key findings: (i) the overall pattern of cross-link sites is remarkably similar between the yeast and the previously described human system, even though transcription initiates downstream of the DNA-TBP-TFIIB-RNAP II-TFIIF complex in the S. cerevisiae system; (ii) the yeast Rpb7 subunit of RNAP II forms strong and reproducible cross-links to both strands of promoter DNA; and (iii) a TFIIB arginine-78 to cysteine replacement (R78C), which shifts start site selection downstream of normal, does not affect TFIIB-DNA cross-links prior to promoter melting but instead affects downstream TFIIF-DNA interactions. These results support the premise that the overall structure of the RNAP II preinitiation complex is similar in all eukaryotes and imply that yeast RNAP II is able to scan template DNA downstream of the preinitiation complex for acceptable start sites. The novel Rpb7-DNA contact sites imply that either promoter DNA does not follow a straight path from TATA to the initiation site or the topology of Rpb7 within the DNA-TBP-TFIIB-RNAP II-TFIIF complex is different from that defined in the 12-subunit RNAP II X-ray structure. We discuss the implications of these results for the mechanism of preinitiation complex assembly and promoter melting.
Chen
Bo-Shiun
BS
Department of Biochemistry, Division of Nucleic Acids Enzymology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Mandal
Subhrangsu S
SS
Hampsey
Michael
M
D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010777:Photochemistry; D011401:Promoter Regions, Genetic / Q000235:genetics; D012319:RNA Polymerase II / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D035181:TATA-Box Binding Protein / Q000378:metabolism; D035581:Transcription Factor TFIIB / Q000378:metabolism; D019473:Transcription Factors, TFII / Q000378:metabolism*; D014158:Transcription, Genetic
D003432:Cross-Linking Reagents; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D019473:Transcription Factors, TFII; D004247:DNA; D001120:Arginine; D012319:RNA Polymerase II; C056604:transcription factor TFIIF