News and Publications

Structural Studies of Complexes Involved in Eukaryotic Transcription and Its Regulation

F.J. Asturias, J.A. Davis, J. Craighead, W.H. Chung, H. Stark

Our research focuses on 3-dimensional electron microscope studies of macromolecular complexes involved in gene expression and the control of transcription. The main player is RNA polymerase II, which is responsible for synthesis of all mRNA in eukaryotes. Polymerase and the general transcription factors form the basal transcription machinery. Control of this machinery is mediated by remodeling of chromatin structure and by regulation of DNA transcription by RNA polymerase II. We study the structure of (1) complexes formed by RNA polymerase II and general transcription factors, (2) coactivator complexes that act as the interface between the basal transcription machinery and activator and repressor proteins that respond to DNA regulatory sequences, and (3) complexes that use energy from ATP to alter chromatin structure, making chromatin accessible for transcription.

A recently determined 3-dimensional structure of RNA polymerase II that includes 2 subunits (Rpb4 and Rpb7, essential for the initiation of transcription) not present in the published x-ray structure of the enzyme reveals that in solution the polymerase exists in 2 conformations. The conformations differ by the position of a "flap" that controls access to the enzyme's DNA-binding channel and active site (Fig. 1). The structure also shows the location of several short amino acid sequences not detected in the x-ray map, some of which may play a crucial role in the synthesis of mRNA. A 20-Å-resolution structure of a complex formed by RNA polymerase II and transcription factor IIF, a factor essential for initiation and elongation, reveals significant conformational changes in the polymerase. Interpretation of these changes will greatly add to our understanding of transcription. Complexes between RNA polymerase II and other general transcription factors are also under study.

Late last year, we published the first low-resolution structures of 3 Mediator coactivator complexes isolated from yeast, mouse, and human cells (Fig. 2). Structural similarities are apparent between the complexes and extend to the "holoenzyme" complexes the Mediator structures form on species-specific interaction with RNA polymerase II, suggesting that Mediator complexes may have a common mechanism of action. Characterizing the structure of the RNA polymerase holoenzyme and the way in which it is affected by activators and repressors is essential for understanding the mechanism of regulation. We determined a 3-dimensional structure of the yeast RNA polymerase II holoenzyme in which an extended Mediator complex envelops the polymerase and makes several contacts with it, while at the same time leaving most of the surface of the polymerase accessible for interaction with other components of the transcription initiation complex. We are determining a precise docking of the structure of RNA polymerase II within the structure of the holoenzyme and are mapping the location of Mediator subunits.

PUBLICATIONS
Dotson, M.R., Yuan, C.X., Roeder, R.G., Myers, L.C.,Gustafsson, C.M., Jiang, Y.W., Li, Y., Kornberg, R.D., Asturias, F.J. Structural organization of yeast and mammalian Mediator complexes. Proc. Natl. Acad. Sci. U. S. A. 97:14307, 2000.