The long-term goal of our research is to understand transcription mechanisms of cellular RNA polymerase (RNAP) and its regulation. During the next project period, we will study the transcription machinery in bacteria to provide a fundamental mechanism of transcription, which is conserved from bacteria to human. Recently, we reported the first X-ray structure of the Escherichia coli RNAP s70 holoenzyme. This enzyme is the most studied RNAP and has been used as a model RNAP for understanding the mechanism of transcription. E. coli RNAP is conveniently prepared using an overexpression system, which allows exploring new directions in RNAP structural studies including the transcription elongation complex, paused transcription complex, RNAP in complex with a variety of transcription factors and inhibitors, as well as RNAP mutants. Here, we propose structural and biochemical studies of E. coli RNAP transcription to address three specific aims.
Aim1. Structural basis for productive-phase transcription: We crystallized an E. coli RNAP elongation complex and determined its X-ray structure at 6 resolution, which provides a framework for the structure- based study of the transcription mechanism. Further experiments are proposed: (1) to determine the atomic resolution structure of the elongation complex for analyzing interactions between RNAP and nucleic acids; (2) to determine structures of the elongation complex with elongation factors NusG or RfaH for determining the positions of the non-template DNA in the transcription bubble and the upstream DNA for the first time in the context of an intact elongation complex; and (3) to determine the structure of the elongation complex with an RNAP mutant prone to transcription slippage for understanding transcriptional fidelity. Our E. coli RNAP elongation complex crystal can extend multiple RNA bases in crystal form. Therefore, we will carry out in crystallo transcription and record motions of the bridge helix and trigger loo as well as translocation of nucleic acids during transcription elongation using time-resolved soak-trigger-freeze X-ray crystallography.
Aim 2. Elucidate the molecular mechanism of transcription pausing by the pause-trigger sequence: Nascent transcript sequencing of the E. coli transcriptome identified a consensus pause sequence in the E. coli genome. We will determine the crystal structure of the elongation complex containing the consensus pause sequence to reveal the interplay between RNAP and nucleic acids during transcription pausing and to provide novel insight into gene regulation.
Aim 3. Structural basis for RNAP modulation by NusA: Most transcription elongation complexes in vivo associate with NusA, which stimulates the effect of RNA hairpins for pausing and termination. We will determine the crystal structures of NusA in complex with RNAP and also with the elongation complex to elucidate the structural basis for NusA-dependent pausing and termination.

Public Health Relevance

E. coli RNAP is the most studied bacterial RNAP and has been used as the model RNAP for screening and evaluating potential RNAP-targeting antibiotics. Because the sequence and antibiotic sensitivity of E. coli RNAP are similar to those of pathogen-related RNAPs, including Mycobacterium tuberculosis and Staphylococcus aureus, E. coli RNAP can now be used to readily study RNAP-antibiotic interactions by X-ray crystallography.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Sledjeski, Darren D
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Pennsylvania State University
Schools of Arts and Sciences
University Park
United States
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Narayanan, Anoop; Vago, Frank S; Li, Kunpeng et al. (2018) Cryo-EM structure of Escherichia coli ?70 RNA polymerase and promoter DNA complex revealed a role of ? non-conserved region during the open complex formation. J Biol Chem 293:7367-7375
Molodtsov, Vadim; Sineva, Elena; Zhang, Lu et al. (2018) Allosteric Effector ppGpp Potentiates the Inhibition of Transcript Initiation by DksA. Mol Cell 69:828-839.e5
Molodtsov, Vadim; Murakami, Katsuhiko S (2018) Minimalism and functionality: Structural lessons from the heterodimeric N4 bacteriophage RNA polymerase II. J Biol Chem 293:13616-13625
Mosaei, Hamed; Molodtsov, Vadim; Kepplinger, Bernhard et al. (2018) Mode of Action of Kanglemycin A, an Ansamycin Natural Product that Is Active against Rifampicin-Resistant Mycobacterium tuberculosis. Mol Cell 72:263-274.e5
Sutherland, Catherine; Murakami, Katsuhiko S (2018) An Introduction to the Structure and Function of the Catalytic Core Enzyme of Escherichia coli RNA Polymerase. EcoSal Plus 8:
Bruhn-Olszewska, Bo?ena; Molodtsov, Vadim; Sobala, Micha? et al. (2018) Structure-function comparisons of (p)ppApp vs (p)ppGpp for Escherichia coli RNA polymerase binding sites and for rrnB P1 promoter regulatory responses in vitro. Biochim Biophys Acta Gene Regul Mech 1861:731-742
Murakami, Katsuhiko S; Shin, Yeonoh; Turnbough Jr, Charles L et al. (2017) X-ray crystal structure of a reiterative transcription complex reveals an atypical RNA extension pathway. Proc Natl Acad Sci U S A 114:8211-8216
Molodtsov, Vadim; Scharf, Nathan T; Stefan, Maxwell A et al. (2017) Structural basis for rifamycin resistance of bacterial RNA polymerase by the three most clinically important RpoB mutations found in Mycobacterium tuberculosis. Mol Microbiol 103:1034-1045
Yakhnin, Alexander V; Murakami, Katsuhiko S; Babitzke, Paul (2016) NusG Is a Sequence-specific RNA Polymerase Pause Factor That Binds to the Non-template DNA within the Paused Transcription Bubble. J Biol Chem 291:5299-308
Gu, Huiqiong; Yoshinari, Shigeo; Ghosh, Raka et al. (2016) Structural and mutational analysis of archaeal ATP-dependent RNA ligase identifies amino acids required for RNA binding and catalysis. Nucleic Acids Res 44:2337-47

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