Topological localization of the carboxyl-terminal domain of RNA polymeraseII 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 C TD along promoter DNA in the transcriptional pre-initiation complex. Compar ison of the promoter contacts made by RNAP II with or without the CTD indic ate 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, i ndicating that the CTD is accessible for protein-protein interactions in a complex containing RNAP II and the general initiation factors. In conjuncti on with previously published observations, our structural data are fully co mpatible with the notion that DNA wrapping around RNAP II places the CTD an d the RNA exit chan nel into juxtaposition and provide a rationale for cont acts between the SRB-mediator complex and core RNAP II observed in the RNAP II holoenzyme.