STRUCTURE DETERMINATION OF RNA POLYMERASE II BY XRAY DIFFRACTION METHOD

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The goal of this project is to determine the X-ray structures of RNA polymerase II and of its complexes with nucleic acids and auxiliary protein factors at atomic resolution. The problem is challenging, since the polymerase alone comprises 15 polypeptides with a total mass of nearly 600,000 Daltons, and addition of the auxiliary factors doubles both the number of polypeptides and the protein mass. Solution of the problem requires a combination of biochemical studies, electron and X-ray crystallography. During the past decade, we have completed the biochemical dissection of the yeast RNA polymerase II transcription system, and we have determined RNA polymerase II structure at 16
resolution by two-dimensional (2D) electron microscopy crystallography. Recently, we have obtained 3-D crystals that diffracted to 3.1
and have thereafter resolved phases to 6.5
using MIRAS method. The result of this project will provide a basis for understanding a vast body of biochemical and getetic data on transcription accumulated in diverse systems over the past 30 years.