A DNA-tethered cleavage probe reveals the path for promoter DNA in the yeast preinitiation complex.
To directly map the position of promoter DNA within the RNA polymerase II (Pol II) transcription preinitiation complex (PIC), FeBABE was tethered to specific sites within the HIS4 promoter and used to map exposed surfaces of Pol II and the general transcription factors in proximity to DNA. Our results distinguish between previously proposed models for PIC structure and demonstrate that downstream promoter DNA is positioned over the central cleft of Pol II, with DNA upstream of TATA extending toward the Pol II subunit Rpb3. Also mapped were segments of TFIIB, TFIIE, TFIIF and TFIIH in proximity to promoter DNA. DNA downstream of the transcription bubble maps to a path between the two helicase subdomains of the TFIIH subunit Rad25 (also called XPB). Together, our results show how the general factors and Pol II converge on promoter DNA within the PIC.
Miller
Gail
G
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N., Seattle, Washington 98109, USA.
Hahn
Steven
S
D001483:Base Sequence; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004257:DNA Polymerase II / Q000737:chemistry / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics*; D017665:Hydroxyl Radical; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051758:Transcription Factor TFIIH / Q000737:chemistry / Q000235:genetics; D014158:Transcription, Genetic
D004271:DNA, Fungal; C489575:SSL2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D051758:Transcription Factor TFIIH; D017665:Hydroxyl Radical; D004257:DNA Polymerase II; D004265:DNA Helicases