Transcription is the major control point of gene expression and RNA polymerase (RNAP), conserved from bacteria to man, is the central enzyme of transcription. Our long term goal is to understand the mechanism of transcription and its regulation. Determining three-dimensional structures of RNAP and its complexes with DNA, RNA, and regulatory factors, is an essential step. We focus on highly characterized prokaryotic RNAPs. The basic elements of the transcription cycle, initiation, elongation, and termination, were elucidated through study of prokaryotes. A detailed structural and functional understanding of the entire transcription cycle is essential to explain the fundamental control of gene expression and to target RNAP with small-molecule antibiotics. Advances in this understanding are stuck on the difficulty of visualizing transient intermediates that underlie the key transition between stable states of the transcription cycle, and the difficulty of visualizing complex macromolecular assemblies involved in regulation, structural problems where X-ray crystallography has severe limitations. While the stable RNAP states around the transcription cycle (RNAP catalytic core, RNAP holoenzyme, RNAP holoenzyme open promoter complex, RNAP elongation complex) are relatively well characterized and understood, the transitions between the stable states are poorly understood. Major transitions include: Holoenzyme + promoter DNA ? open promoter complex (initiation) Open promoter complex > elongation complex (promoter escape, ? dissociation) Elongation complex > core RNAP + DNA + completed RNA transcript (termination) Each of these transitions are characterized by unstable, transient intermediates that are extremely challenging for structural biology. At every stage of the transcription cycle, RNAP function is modulated by interactions with extrinsic regulatory factors. Assembling and crystallizing transcription complexes containing extrinsic regulators also presents challenges for structural biology. Due to recent advances, cryo-electron microscopy (cryo-EM) now offers a route to structural and mechanistic characterization of these intermediates and large assemblies. We will use cryo-electron microscopy, in combination with X-ray crystallography and other approaches, to exploit this opportunity and provide a complete characterization of the bacterial transcription cycle.

Public Health Relevance

We focus on highly characterized bacterial RNA polymerases, which have a high degree of conservation of structure and function from bacteria to man. The bacterial RNA polymerase is a proven target for antimicrobials, such as rifampicin (or its derivatives), widely used in combination therapy to treat tuberculosis, but bacterial strains resistant to rifampicin arise with appreciable frequency, compromising treatment. Insights into the mechanism of bacterial transcription, as well as into the inhibition mechanism of inhibitors, can lead to new avenues for the development of antimicrobials.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Unknown (R35)
Project #
1R35GM118130-01
Application #
9071516
Study Section
Special Emphasis Panel (ZGM1)
Program Officer
Flicker, Paula F
Project Start
2016-05-01
Project End
2021-04-30
Budget Start
2016-05-01
Budget End
2017-04-30
Support Year
1
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Rockefeller University
Department
Physiology
Type
Graduate Schools
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Kang, Jin Young; Mooney, Rachel Anne; Nedialkov, Yuri et al. (2018) Structural Basis for Transcript Elongation Control by NusG Family Universal Regulators. Cell 173:1650-1662.e14
Peek, James; Lilic, Mirjana; Montiel, Daniel et al. (2018) Rifamycin congeners kanglemycins are active against rifampicin-resistant bacteria via a distinct mechanism. Nat Commun 9:4147
Boyaci, Hande; Chen, James; Lilic, Mirjana et al. (2018) Fidaxomicin jams Mycobacterium tuberculosis RNA polymerase motions needed for initiation via RbpA contacts. Elife 7:
Kang, Jin Young; Mishanina, Tatiana V; Bellecourt, Michael J et al. (2018) RNA Polymerase Accommodates a Pause RNA Hairpin by Global Conformational Rearrangements that Prolong Pausing. Mol Cell 69:802-815.e1
Chen, James; Wassarman, Karen M; Feng, Shili et al. (2017) 6S RNA Mimics B-Form DNA to Regulate Escherichia coli RNA Polymerase. Mol Cell 68:388-397.e6
Kang, Jin Young; Olinares, Paul Dominic B; Chen, James et al. (2017) Structural basis of transcription arrest by coliphage HK022 Nun in an Escherichia coli RNA polymerase elongation complex. Elife 6:
Feklistov, Andrey; Bae, Brian; Hauver, Jesse et al. (2017) RNA polymerase motions during promoter melting. Science 356:863-866
Campbell, Elizabeth A; Kamath, Shreya; Rajashankar, Kanagalaghatta R et al. (2017) Crystal structure of Aquifex aeolicus ?N bound to promoter DNA and the structure of ?N-holoenzyme. Proc Natl Acad Sci U S A 114:E1805-E1814
Wang, Guanshi; Hauver, Jesse; Thomas, Zachary et al. (2016) Single-Molecule Real-Time 3D Imaging of the Transcription Cycle by Modulation Interferometry. Cell 167:1839-1852.e21