Before the genetic information stored in the DNA code can be utilized it must be transcribed, in a process in which an RNA copy of the DNA code is made. Our research focuses on structural studies of macromolecular complexes involved in eukaryotic gene expression. Control of a gene expression is mostly effected through the regulation of DNA transcription, which is therefore of utmost significance in cell differentiation and development, and in the response of living cells to their environment. Complex molecular machines comprising many individual proteins perform specific steps of the transcription process. At the center of this complex apparatus is RNA polymerase II (RNAPII), the enzyme responsible for reading the DNA information and synthesizing an RNA copy. In order to carry out transcription and respond to regulatory signals, RNAPII must interact with many other molecules. The objective of our research is to reveal the mechanisms of transcription and its regulation. Electron microscope (EM) images of macromolecular complexes are analyzed to reconstruct their three-dimensional structures, as they appear under conditions similar to those in a living cell. The end result of these efforts will be an understanding of the mechanism of transcription at the molecular level.

RNA polymerase II (RNAPII) is the center piece of the basal transcription machinery. Roger Kornberg and his group started by carrying out 2-D electron crystallograpy studies, and eventually characterized a 10-subunit form of the enzyme to atomic resolution by X-ray crystallography. EM studies first identified the location of the two subunits absent in the original X-ray structures. However, the most important contribution from EM has been an understanding of the conformation of the enzyme in solution, which had important implications for understanding the way in which RNAPII first interacts with promoter DNA. Characterization of RNAPII by EM also paved the way for interpretinf EM structures of larger complexes including basal transcription factors and the Mediator complex.

The general transcription factor TFIIF (comprising three different polypeptides, Tfg1, Tfg2, and Tfg3) is critically involved in mediating the interaction of RNAPII with promoter DNA. In particular, the middle subunit of TFIIF, Tfg2, has a significant degree of sequence and functional homology to the bacterial sigma factor. We determined the structure of the RNAPII/Tfg2 complex, as well as that of the entire RNAPII/TFIIF complex. Binding of TFIIF triggers a number of large-scale conformational changes in RNAPII. By considering these changes along with the RNAPII/TFIIF structure, we were able to map the distribution of TFIIF density, and map the distribution of the domains corresponding to Tfg2. Our results suggest that Tfg2 is a structural homolog of the bacterial sigma factor and is likely to play a role in directing promoter DNA to the active site cleft of RANPII.

Transcription initiation requieres the assembly of a large multi-component complex in which RNAPII interacts with a number of accessory proteins known as the general transcription factors (GTFs) to form the preinitiation complex (PIC). The PIC, including nearly 30 different polypeptides, assembles at a promoter and allows RNAPII to recognize the transcription start site. One of the most ambitious goals of our research is to obtain a structure of the entire PIC. A subset of the PIC components including RNAPII, TFIIF, the TATA binding protein (TBP), and TFIIB, constitute the minimum assembly capable of promoter-dependent initiation. We proposed a model for organization of this minimal PIC based on the structure of the RNAPII/TFIIF complex, and work to obtain a structure the minimal PIC is currently underway.

Mediator, a large complex comprising over 25 different polypetide subunits, is arguably the key player in regulation of transcription in all eukaryotic organisms. Mediator was first characterized in yeast cells, but Mediator homologs have now been identified in all eukaryotes examined. We have generated 3-D structures of yeast, mouse, and human Mediators, as well as a structure of the yeast Mediator/RNAPII (holoenzyme) complex. One of the most intriguing aspects of Mediator structure is that it undergoes a dramatic conformational change upon interaction with RNAPII. This conformational change might play a central role in the regulation mechanism through its effect on the assembly of the PIC.

A variety of activators and repressors interact directly with Mediator which then works as the interface that conveys regulatory information to the basal transcription machinery. Characterization of the Mediator/RNAPII holoenzyme revealed their relative orientation, with Mediator interacting with the back face of polymerase. This is homologous to what has been observed in bacterial cells (where regulatory proteins also interact with polymerase subunits located behind the upstream end of the active site cleft) and suggest the possible conservation of fundamental aspects of the regulation mechanism. While the specific mechanism of regulation by Mediator remains unknown, it seems likely that Mediator might exert an effect on assembly of the PIC.

Presented here is a possible mechanism for transcription regulation by Mediator. In this picture, Mediator would play a key role in promoting assembly of the PIC, a process that in turn would be affected by the conformational state of Mediator, possibly controlled by its interaction with activators and repressors. Once initial assembly of the PIC was completed and the initial round of transcription started, additional rounds of transcription might be facilitated by a Scaffold complex (biochemically characterized by others) that would remain at the promoter, thereby speeding up assembly of a new PIC. This mechanism is only speculative, and further biochemical and structural work will be required to test it.

Structural characterization of the eukaryotic transcription machinery remains the central goal of our research efforts. Cryo-EM studies of yeast and human complexes are currently underway. We are also complementing our EM efforts with biochemical and functional studies preinitiation complex assembly in yeast. We are interested in complementary approaches, such as single-molecule techniques, that could provide additional insight into the mechanism of transcription regulation.