Heads of Laboratories
Seth A. Darst, Ph.D.
Jack Fishman Professor
Laboratory of Molecular Biophysics
RNA, the blueprint for proteins, is made in all cells by a complex molecular machine, the DNA-dependent RNA polymerase. Dr. Darst’s research explores the mechanism and regulation of transcription by determining three-dimensional structures of RNA polymerase and associated proteins. The RNA polymerase is conserved from bacteria to humans, making the simpler bacterial version an excellent model for understanding how transcription and, by extension, gene expression are controlled.
In its simplest bacterial form, the RNA polymerase complex is made up of at least four proteins, while the eukaryotic complex comprises a dozen individual proteins or more. However, the catalytic core of RNA polymerase is evolutionarily conserved among all organisms, with a very high retention of the basic sequence. In all cells, the RNA polymerase core can synthesize RNA chains from a DNA template but is unable to recognize promoters, specific sites within DNA where transcription initiates. 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.
The Darst laboratory is purifying, crystallizing and determining the structures of proteins involved in transcription using a combination of approaches, including x-ray crystallography and 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. The results were 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 move 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, Dr. 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.
Research from the Darst lab has also described the structure of the σ protein, the key transcription initiation factor in bacteria. Another structural study by members of the lab 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.
Dr. 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.
Dr. Darst received his B.S. in chemical engineering from the University of Colorado, Boulder, in 1982. He received his M.S. in chemical engineering in 1984 and his Ph.D. in chemical engineering in 1987, both from Stanford University. Remaining at Stanford, Dr. Darst was an American Cancer Society Postdoctoral Fellow from 1987 to 1990 and a Lucille P. Markey Postdoctoral Scholar from 1990 to 1992. He came to Rockefeller in 1992 as assistant professor and became associate professor in 1997 and professor in 2000.
Dr. Darst was named a Pew Scholar in the Biomedical Sciences in 1995 and a Career Scientist of the Irma T. Hirschl Charitable Trust in 1994. He is a member of the National Academy of Sciences.
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.
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