News and Publications

Structural Studies of the Eukaryotic Transcription Machinery

Before the genetic information stored in DNA 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. Many individual proteins associate to form a complex molecular machinery that carries out transcription. At the heart of the system is RNA polymerase II (RNAPII), the enzyme responsible for synthesizing all messenger RNA.

The goal of our research is to reveal the mechanisms 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.

We used the structure of the initiation-competent form of RNAPII, which we published last year, as the starting point for characterization of a complex that includes RNAPII and the general transcription factor IIF (Fig. 1). Factor IIF plays an essential role in modulating the interaction of RNAPII with promoter DNA at initiation and is also important during the elongation phase of transcription. Analysis of the structure of the RNAPII-IIF complex and comparison with the structure of a bacterial initiation complex revealed a similar topology. This unexpected result underscores the conservation of the basic mechanism of transcription initiation between bacteria and higher cells.

By combining information from the RNAPII-IIF structure with the previously published structure of a complex formed by DNA and 2 additional transcription factors, IIB and TBP, we could, for the first time, propose a model for the organization of the catalytic core of the eukaryotic transcription machinery, comprising RNAPII, IIF, IIB, TBP, and DNA (Fig. 2). The model predicts the path that promoter DNA follows along the surface of RNAPII during initiation. Upstream promoter DNA travels across a patch on the surface of RNAPII that is central for interaction of the enzyme with Mediator, a large multiprotein complex that conveys regulatory information to the basal transcription machinery. The corresponding area on the surface of the bacterial polymerase also plays a role in regulation, once more pointing to a high degree of conservation of all aspects of transcription in all living organisms. This result also suggests a structural explanation for the role that Mediator plays in the reinitiation of transcription in highly transcribed genes. The role of Mediator in regulation may be closely linked to its effect on RNAPII recruitment and on stabilization of the transcription preinitiation complex.

We will confirm our predictions about the organization of the catalytic core of the transcription machinery by calculating the actual structure of the core, and we will continue to build the entire transcription apparatus by adding the 2 final components, transcription factors IIE and IIH. Inclusion of the Mediator complex will eventually reveal the details of its interaction with the basal transcription machinery. We are also characterizing the human transcription machinery. A 15-Å resolution of human RNAPII confirmed the expected structural similarity of the polymerase to the yeast enzyme but also revealed important differences in the active site, differences that we might be able to correlate to known differences in the organization of yeast and mammalian promoters. Finally, we continue to work on the structural characterization of complexes that affect transcription regulation by affecting the structure of chromatin.

Publications

Asturias, F.J. RNA polymerase II and the transcription preinitiation complex. Bioessays, in press.

Asturias, F.J., Craighead J.L. RNA polymerase II at initiation. Proc. Natl. Acad. Sci. U. S. A., in press.

Asturias, F.J., Ezeokonkwo, C., Kornberg, R.D., Lorch, Y. Electron microscopic analysis of the RSC chromatin remodeling complex. Methods Enzymol., in press.

Chung, W.H., Craighead, J.L., Chang, W.H., Ezeokonkwo, C., Bareket-Samish, A., Kornberg, R.D., Asturias, F.J. RNA polymerase II/TFIIF structure and conserved organization of the initiation complex. Mol. Cell, in press.