Description of Research Expertise

Transcription initiation by RNA polymerase II (pol II) is the first step in gene expression, and an end point of many signal transduction pathways. Approximately sixty proteins assemble in a 3-million Dalton complex at all mRNA promoters before every round of transcription. About half of these proteins, some 30 in number [the subunits of pol II and the general transcription factors (GTFs; TBP, TFIIA, TFIIB, TFIIE, TFIIF, TFIIH)], form a pre-initiation complex (PIC) that can recognize a promoter, open the double-stranded DNA, select a transcription start site, and synthesize a nascent transcript (Fig. 1). The remaining proteins are needed for the regulation of transcription. Emerging evidence from recent genome-wide studies points to the transition from initiation to elongation as the most widespread regulatory mechanism of transcription. In humans, this transition is the basis for regulation during environmental stress, immunological signaling and development, and is targeted by a variety of transcription factors (e.g., DSIF, NELF, P-TEFb, mRNA capping enzyme) (Fig. 1). Therefore, the elucidation of this mechanism of action is critical for finding novel targets for cancer therapies.



Figure 1. A model for the transition from initiation to elongation of RNA polymerase II.

Previous studies were performed with nuclear extracts or with partially purified GTFs assembled on immobilized promoter DNA. Due to the poor efficiency of the reaction and trace amounts of protein involved, detection was only possible by the synthesis of a radiolabeled transcript and by immunoblotting techniques. In an important advance during my postdoctoral work with Dr. Roger Kornberg at Stanford University, we overcame this limitation by succeeding in reconstituting milligram quantities of a complete 32-subunit PIC that included all the GTFs and pol II. This has more than 30-fold greater transcription activity than that obtained by simple mixing of components, as routinely done in the past (Murakami et al., 2012, PNAS, Murakami et al., 2013, JBC; Murakami et al., 2015, in press, Mol. Cell). It opened the way to definitive biochemical and structural studies of the entire transcription initiation machinery. In the first of such studies, we have determined the structure of the PIC at the closed state by cryo-electron microscopy (cryo-EM) at 15 Å resolution (Murakami et al., 2013, Science) and subnanometer resolution (Murakami et al., 2015, Manuscript in preparation). The way is now open to dissect the post-initiation process by single-molecule studies and to determine the structures of post-initiation intermediates by cryo-electron microscopy (cryo-EM).