E.coli RNA Polymerase: The Darst Lab

RNA in all cellular organisms is synthesized by a complex molecular machine, the DNA-dependent RNAP. In its simplest bacterial form, the enzyme comprises four subunits with a total molecular mass of ~400 kDa. We focus on highly characterized prokaryotic RNAPs, which share basic structure and catalytic function with more complex archaeal and eukaryotic enzymes but are controlled by a much simpler set of regulatory factors.

We investigate the basic elements of the transcription cycle, initiation, elongation, and termination, elucidated through study of the model organism Escherichia coli. The transcription cycle is marked by a series of stable RNAP complexes (core → holo → RPo → EC) that interconvert through transient intermediates involving large conformational changes in the nucleic acids, the RNAP, or both. At every stage of the transcription cycle, RNAP function is modulated by interactions with hundreds of extrinsic regulatory factors. Moreover, bacteriophage have evolved extrinsic factors that use ingenious mechanisms to subvert the host transcription process for their own purposes. We use a combination of biochemical and biophysical approaches, with an emphasis on cryo-electron microscopy, to fill the gaps in our understanding of the bacterial transcription cycle, particularly large regulatory complexes and unstable transition states between the stable states of the cycle.

Transcription of Mycobacterium tuberculosis: The Campbell Lab ​

RNA polymerase (RNAP), the enzyme responsible for bacterial transcription, is a proven and effective target of antimicrobials in many pathogens, including the causative agent of tuberculosis (TB), Mycobacterium tuberculosis. We investigate the mechanisms and regulation of the transcriptional process and the molecular interactions between the RNAP and its inhibitors using cryo-EM and other tools.

Our goals are two-pronged: 1) to provide structural and functional insights to guide TB drug discovery and optimization and 2) to shed light on the fundamental process of transcription. We are highly collaborative, working in conjunction with other investigators from other universities and Rockefeller to investigate new aspects of how antibiotics and transcription factors modulate the RNAP from Mycobacterium tuberculosis. To accomplish this, we use an array of biochemical, molecular, microbial, and biophysical approaches, including cryo-electron microscopy, real-time fluorescence kinetic studies, antibiotic inhibition assays, and genetic screens. In addition, we have recently started to explore similar aspects of RNAP from another deadly pathogen, Clostridium difficile.  
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SARS-CoV-2: The Darst Lab and the Campbell Lab

SARS-CoV-2 is the causative agent of the current COVID-19 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (holo-RdRp, subunits nsp7/nsp82/nsp12) in a replication-transcription complex (RTC), which is the target for antivirals such as remdesivir and molnupiravir. In addition to its essential replication-transcription activity, nsp12 contains an N-terminal nidovirus specific domain, know as the NiRAN domain, with unknown function, but with enzymatic activity that is also essential for viral propagation. The holo-RdRp is thought to coordinate with a cast of additional factors to carry out its function. However, the nature of these interactions has not been elucidated.
 
In Spring 2020, in light of the pandemic, we combined efforts with other PIs at the Rockefeller University (Drs. Chait and Kapoor) to study the replication and “transcription” complex of SARS-CoV-2. Our goal is to identify regulators of this process using binding assays and native mass-spectrometry to identify factors that regulate and interact with the RdRp. We have shown that the helicase nsp13 makes a stable complex with the holo-RdRp and resolved structures of this complex by cryo-EM. These structures have led to structure-based models of RTC backtracking, template switching and replication which we are currently exploring. In addition, we are trying to work out the other processes the virus uses to express its ORFs and to replicate its genome.