Structural Determination of a Transcribing RNA Polymerase II Complex.

The purpose of the proposed research is to provide a structural basis for understanding the mechanism of transcription, its regulation, and altered regulation as occurs in tumor cells. The goal of the proposed research is to determine the X-ray structure of RNA Polymerase II in the midst of transcribing RNA from a DNA template. During the first year of funding, template DNA was shortened to a minimal size without affecting the stability of the paused complex or its efficiency of formation. It was determined that previous platelike crystals are sensitive to physical manipulation, soaking in cryoprotectant or soaking with heavy metal clusters. Crystallization trials of polymerase resulted in new non-platelike crystal forms allowing for the collection of a preliminary data set to 7.5 Angstroms. This is the best elongation complex data set, since previous platelike crystals had mosaic spreads over 1 degree. Recently new crystallization conditions allowed the growing of crystals in cryoprotectant and the generation of yet new crystal forms. Growing in cryoprotectant is a major accomplishment considering the sensitivity of the crystals to manipulations.