Structure of RNA Polymerase II

Stanford University, Stanford CA

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Abstract

DESCRIPTION (provided by applicant): The long range goal of our research is the understanding of RNA polymerase II (pol II) transcription in all its aspects: initiation, RNA chain elongation, and control of the process. Specific aims for the next project period are as follows: (1) Extension of resolution of the pol II transcribing complex structure. Preliminary results have shown the possible path of the pol II C- terminal domain (CTD), the likely role of water molecules in the protein-nucleic acid translocation process, and a rotation of the penultimate base in the DNA template that may pair with NTP prior to entry in the active center (templated NTP). (2) Determination of pre-initiation complex (PIC) structure. After many failed attempts at forming complexes of pol II with individual general transcription factors (GTFs), we succeeded in the assembly of a complete PIC, containing pol II and all six GTFs. This remarkable advance has opened the way to structural studies of the transcription initiation process. We propose protein-protein cross- linking and mass spectrometry (with the use of a novel ion source), soaks of pol II crystals with peptides from GTFs, and docking of crystal structures of GTFs to the peptide-pol II cocrystal structures. (3) Determination of Mediator structure. We propose to extend our structural analysis of the 7-subunit 220 kDa Mediator Head module, currently at 4.3 A, to higher resolution and to the complete 21-subunit 1 MDa Mediator complex.