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Structural Studies of the Eukaryotic Transcription Machinery

F.J. Asturias, J.A. Davis, J.L. Craighead, W.H. Chung, H. Stark, C. Ezeokonkwo, I. Cheung

Before the genetic information stored in the DNA code can be used, it must be transcribed; that is, an RNA copy of the DNA code must be made. Most of the control and regulation of gene expression is focused on this transcription process, which is therefore of utmost importance in cell differentiation and development and in the response of all living cells to their environment. A complex molecular machinery, composed of many individual proteins that associate to form complexes that perform specific steps of the process, carries out transcription. At the heart of this complex apparatus is RNA polymerase II, the enzyme responsible for reading the DNA information and synthesizing an RNA copy. To carry out transcription and respond to regulatory signals, the polymerase must interact with many other molecules.

The goal of our research is to reveal the mechanism of transcription and its regulation by studying the structure of the different components of the transcription machinery and the complexes that they form. We use electron microscopy to reconstruct the 3-dimensional structures of the complexes as the structures appear under conditions similar to those in a living cell. The result of these efforts will be an understanding of the mechanism of transcription at the molecular level.

Structural characterization of RNA polymerase II suggests a role for Rpb7, an essential polymerase subunit, in delivering newly synthesized mRNA to the mRNA-processing machinery. The conformation of the active-site cleft of the polymerase has resulted in a revised model for interaction of the enzyme with its promoter DNA substrate (Fig. 1). Efforts to determine the structures of complexes formed by RNA polymerase II and the general transcription factors continue. The findings will contribute to our understanding of how the components of the basal transcription machinery are assembled to initiate transcription.

We recently determined the precise orientation of RNA polymerase II in the holoenzyme complex formed by the polymerase and Mediator, a multiprotein complex that is essential to convey regulatory information to the polymerase. Characterization of the interaction between RNA polymerase II and Mediator provides a structural basis for understanding the way in which the response to enhancer and suppressor DNA sequences is enabled and is used to regulate the function of the polymerase and control gene expression. Interaction of RNA polymerase II with Mediator centers on the Rpb3-Rpb11 polymerase subunit complex, the eukaryotic homolog of the bacterial aa´ homodimer involved in the regulation of transcription in prokaryotes (Fig. 2). This part of the polymerase structure may be a conserved surface essential for transduction of regulatory information to the enzyme.

PUBLICATIONS

Asturias, F.J., Chung, W., Kornberg, R.D., Lorch, Y. Structural analysis of the RSC chromatin-remodeling complex. Proc. Natl. Acad. Sci. U. S. A. 99:13477, 2002.

Craighead, J.L., Chang, W.H., Asturias, F.J. Structure of yeast RNA polymerase II in solution: implications for enzyme regulation and interaction with promoter DNA. Structure (Camb.) 10:1117, 2002.

Davis, J.A., Takagi, Y., Kornberg, R.D., Asturias, F.J. Structure of the yeast RNA polymerase II holoenzyme: Mediator conformation and polymerase interaction. Mol. Cell 10:409, 2002.