Seth A. Darst, Ph.D.
Jack Fishman Professor
All cells make RNA, the blueprint for proteins, with a complex molecular machine called the DNA-dependent RNA polymerase. Using bacteria as a model organism, Darst’s group determines three-dimensional structures of RNA polymerase and associated proteins in order to explore the mechanism and regulation of transcription. This work has implications for understanding how gene expression is controlled in many organisms.
In its simplest bacterial form, the DNA-dependent RNA polymerase (RNAP) comprises four subunits with a total molecular mass of approximately 400 kDa. The Darst lab focuses on highly characterized prokaryotic RNAPs, which share basic structure and catalytic function with more complex archaeal and eukaryotic enzymes but are controlled by a simpler set of regulatory factors.
Through the study of prokaryotes, scientists have elucidated the basic steps of the transcription cycle: initiation, elongation, and termination. This cycle begins when the RNAP catalytic core combines with initiation factors (called σ factors in bacteria) to generate the active RNAP holoenzyme. RNAP then locates promoter sequences within duplex DNA, forms the open promoter complex (RPo) by unwinding the DNA surrounding the transcription start site, initiates the synthesis of an RNA chain, and elongates the RNA processively in an elongation complex (EC) while translocating itself and the transcription bubble along the DNA template. Then, finally, it releases itself and the completed RNA transcript from the DNA when it encounters termination signals.
The Darst lab uses a combination of approaches to understand the structure, function, and regulation of the entire transcription cycle. The lab has used X-ray crystallography to determine structures of stable RNAP complexes that mark the transcription cycle (the RNAP core enzyme, holoenzyme, open promoter complex, and elongation complex). More recently, the Darst group has used cryo-electron microscopy to investigate how these complexes interconvert through transient intermediates involving large conformational changes in the nucleic acids, RNAP, or both.
At every stage of the transcription cycle, extrinsic regulatory factors interact with RNAP to modulate its function. Even in “simple” bacteria, more than 100 RNAP regulators have been identified. Moreover, bacteriophage have evolved extrinsic factors that use ingenious mechanisms to subvert the host transcription process for their own purposes. The Darst group studies the structure and function of these regulatory processes at a deep mechanistic level, often in collaboration with leading groups from around the world.
Darst and his colleagues seek a detailed structural and functional understanding of the entire transcription cycle—knowledge that will be essential to explain the fundamental control of gene expression and to target RNAP with small-molecule antibiotics. More fundamentally, Darst’s group pursues a complete understanding of how the cycle is driven by a complex molecular machine that uses binding and chemical energy to effect conformational changes—and how this process is modulated by regulators.
A related research program in the laboratory, led by research associate professor Elizabeth A. Campbell, seeks to understand the transcription cycle of the human pathogen Mycobacterium tuberculosis to aid in the development of new antibiotics. Campbell and Darst are also studying the structural architecture of the SARS-CoV-2 replicase/transcriptase complex, knowledge that could help in the design of novel treatments for COVID-19.
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.