Topological localization of the carboxyl-terminal domain of RNA polymerase II in the initiation complex.
The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) functions at multiple stages of transcription and is involved in the coupling of transcription to pre-mRNA processing. We have used site-specific protein-DNA photocross-linking to determine the position of the CTD along promoter DNA in the transcriptional pre-initiation complex. Comparison of the promoter contacts made by RNAP II with or without the CTD indicate that the CTD approaches promoter DNA downstream of the transcriptional initiation site between positions +16 and +26. Incubation of pre-assembled initiation complexes with antibodies to the CTD prior to UV irradiation led to specific photocross-linking of the IgG heavy chain to nucleotide +17, indicating that the CTD is accessible for protein-protein interactions in a complex containing RNAP II and the general initiation factors. In conjunction with previously published observations, our structural data are fully compatible with the notion that DNA wrapping around RNAP II places the CTD and the RNA exit channel into juxtaposition and provide a rationale for contacts between the SRB-mediator complex and core RNAP II observed in the RNAP II holoenzyme.
Département de biologie, Faculté des sciences, Université de Sherbrooke, Sherbrooke, Québec, J1K 2R1 Canada.
M
M
Douziech
D
D
Forget
J
J
Greenblatt
B
B
Coulombe
D000345:Affinity Labels; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010782:Photolysis; D011401:Promoter Regions, Genetic; D012313:RNA / Q000737:chemistry; D012319:RNA Polymerase II / Q000737:chemistry*; D012322:RNA Precursors / Q000235:genetics; D013950:Thymus Gland / Q000201:enzymology; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D007143:Immunoglobulin Heavy Chains; D012322:RNA Precursors; D014157:Transcription Factors; D012313:RNA; D004247:DNA; D012319:RNA Polymerase II