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Heads of Laboratories

Head shot of Seth Darst
Seth A. Darst, Ph.D.
Jack Fishman Professor
Laboratory of Molecular Biophysics
Seth.Darst@rockefeller.edu

RNA, the blueprint for proteins, is made by a complex molecular machine, the DNA-dependent RNA polymerase, present in all cells. Using bacteria as a model organism, Darst’s research explores the mechanism and regulation of transcription by determining three-dimensional structures of RNA polymerase and associated proteins. This work has implications for understanding how gene expression is controlled in many organisms.

In its simplest bacterial form, RNA polymerase is made up of at least five proteins, while the eukaryotic complex comprises a dozen or more individual proteins. However, the catalytic core of RNA polymerase is evolutionarily conserved among all organisms, with a very high retention of the basic sequence. The simpler bacterial system can hence provide an important model for investigating transcription as it occurs in all organisms.

The Darst lab is determining the structures of bacterial RNA polymerase and its associated proteins using a combination of approaches, including x-ray crystallography and cryo-electron microscopy. Solving these structures provides scientists with snapshots of different, kinetically stable states of the transcription complex, offering insight into the dynamic events that occur during transcription. As more structures are solved, they will shed light on the molecular mechanisms of the regulatory factors acting at different stages of transcription.

Members of the Darst lab have purified, crystallized, and determined the structure of the core RNA polymerase from the thermophilic eubacteria Thermus aquaticus, producing the first high-resolution structure of a multisubunit cellular RNA polymerase. The lab then tracked the path of the transcript RNA and the template DNA through the polymerase structure using RNA-protein and DNA-protein cross-links, a method that gave the scientists a model of the elongation complex that makes RNA as the polymerase moves along the DNA.

The lab has also worked to understand how the introduction of specific molecules impacts RNA polymerase. To determine how the antibiotic rifampicin inhibits RNA polymerase function, Darst used a combination of x-ray crystallography and biochemical studies. Electron microscopy was used to reveal how a known regulatory factor modulates the transcript elongation process.

All cells must overcome a particular challenge in order to initiate transcription: The RNA polymerase core cannot recognize promoters, specific sites where transcription begins. In bacteria, a separate protein—the σ factor—must bind to the RNA polymerase core to form the RNA holoenzyme, which can locate promoters and open the DNA for transcription. Research from the Darst lab has described the structure of σ, the key transcription initiation factor in bacteria.

Another structural study described the transcription-repair coupling factor, a protein cells use to remove RNA polymerase molecules stalled at sites of DNA damage and to recruit repair proteins to fix the damage.

Darst’s studies have furthered scientists’ knowledge of the transcription process in several ways. Structures of the RNA polymerase holoenzyme and holoenzyme with a promoter DNA fragment have provided insight into transcription initiation. In addition, high-resolution structures of σ factor domains in complex with promoter DNA or with inhibitory anti-σ factors have provided the basis for structural and functional analysis of the key regulatory factor in bacterial transcription.

Darst is a faculty member in the David Rockefeller Graduate Program, the Tri-Institutional M.D.-Ph.D. Program, and the Tri-Institutional Ph.D. Program in Chemical Biology.


EDUCATION

B.S. in chemical engineering, 1982
University of Colorado, Boulder

M.S. in chemical engineering, 1984
Ph.D. in chemical engineering, 1987
Stanford University

POSTDOC

Stanford University, 1987–1992

POSITIONS

Assistant Professor, 1992–1997
Associate Professor, 1997–2000
Professor, 2000–
The Rockefeller University

AWARDS

Irma T. Hirschl/Monique Weill-Caulier Trust Research Award, 1994

Pew Biomedical Scholar, 1995

MEMBERSHIPS

National Academy of Sciences

SELECTED PUBLICATIONS

Kang, J.Y. et al. Structural basis of transcription arrest by coliphage HK022 Nun in an Escherichia coli RNA polymerase elongation complex. eLife 6, e25478 (2017).

Hubin, E.A. et al. Structure and function of the mycobacterial transcription initiation complex with the essential regulator RbpA. Elife 6, e22520 (2017).

Campbell, E.A. et al. Crystal structure of Aquifex aeolicus σN bound to promoter DNA and the structure of σN-holoenzyme. Proc. Natl. Acad. Sci. U.S.A. 114, e1805–e1814 (2017).

Bae, B. et al. Structure of a bacterial RNA polymerase holoenzyme open promoter complex. Elife 4, e08504 (2015).

Bae, B. et al. CarD uses a minor groove wedge mechanism to stabilize the RNA polymerase open promoter complex. Elife 4, e08505 (2015).


Dr. Darst is a faculty member in the David Rockefeller Graduate Program, the Tri-Institutional M.D.-Ph.D. Program, and the Tri-Institutional Ph.D. Program in Chemical Biology.