This proposal addresses transcription initiation, elongation, and termination by bacterial RNA polymerase (RNAP). In transcription initiation, RNAP: (i) binds to promoter DNA, yielding an RNAP-promoter closed complex; (ii) unwinds promoter DNA, yielding an RNAP-promoter open complex; (iii) synthesizes the first -11 nucleotides of RNA as an RNAP-promoter initial transcribing complex, using a scrunching mechanism in which RNAP remains stationary on promoter DNA and pulls in adjacent DNA in each nucleotide-addition cycle; and (iv) escapes from the promoter. In transcription elongation, RNAP synthesizes the remaining nucleotides of RNA as an RNAP-DNA elongation complex, using a stepping mechanism in which RNAP moves forward on DNA in each nucleotide-addition cycle. In transcription termination, RNAP stops synthesizing RNA, releases RNA, and dissociates from DNA. Each of these reactions is a target for regulators. Understanding transcription initiation, elongation, termination, and regulation will require defining the structural transitions in protein and DNA in each reaction, the kinetics of structural transitions, and the mechanisms by which regulators affect structural transitions. In the current period, we defined the structural basis of transcription start-site selection, de nova transcription initiation, non-canonical-initiating-nucleotide-dependent transcription initiation, initial transcription, and Class 11 transcription activation; we developed high-throughput-sequencing approaches that enable comprehensive analysis of DNA-sequence determinants for transcription; we developed multiplexed crosslinking approaches that enable comprehensive analysis of protein-DNA interactions in transcription; we developed ensemble and single-molecule fluorescence assays that enable monitoring of RNAP clamp and RNAP trigger-loop conformation in solution; and we defined binding sites and mechanisms for small-molecule inhibitors of transcription. The proposed work will build on the findings of the current period. The proposed work will use x-ray crystallography, single-molecule biophysics, biochemistry, and genetics to address five specific aims:
Specific Aim 1 : Determination of the structural basis of RNAP slippage Specific Aim 2: Determination of the structural basis of RNAP translocation Specific Aim 3: Analysis of RNAP translocation in elongation, pausing, and termination Specific Aim 4: Analysis of RNAP clamp conformation in elongation, pausing, and termination Specific Aim 5: Analysis of RNAP triqqer-loop conformation in elonqation, pausinq, and termination

Public Health Relevance

Bacterial RNA polymerase (RNAP) is a molecular machine that carries out reactions essential for bacterial gene expression and bacterial growth. Two classes of current antibacterial drugs function by inhibiting RNAP. The proposed work will provide information essential for understanding the mechanism of action of RNAP and for rational design of improved and novel antibacterial drugs that function by inhibiting RNAP.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
Project #
Application #
Study Section
Special Emphasis Panel (NSS)
Program Officer
Adkins, Ronald
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Rutgers University
Organized Research Units
United States
Zip Code
Gabizon, Ronen; Lee, Antony; Vahedian-Movahed, Hanif et al. (2018) Pause sequences facilitate entry into long-lived paused states by reducing RNA polymerase transcription rates. Nat Commun 9:2930
Vvedenskaya, Irina O; Bird, Jeremy G; Zhang, Yuanchao et al. (2018) CapZyme-Seq Comprehensively Defines Promoter-Sequence Determinants for RNA 5' Capping with NAD. Mol Cell 70:553-564.e9
Lin, Wei; Das, Kalyan; Degen, David et al. (2018) Structural Basis of Transcription Inhibition by Fidaxomicin (Lipiarmycin A3). Mol Cell 70:60-71.e15
Duchi, Diego; Mazumder, Abhishek; Malinen, Anssi M et al. (2018) The RNA polymerase clamp interconverts dynamically among three states and is stabilized in a partly closed state by ppGpp. Nucleic Acids Res 46:7284-7295
Sosio, Margherita; Gaspari, Eleonora; Iorio, Marianna et al. (2018) Analysis of the Pseudouridimycin Biosynthetic Pathway Provides Insights into the Formation of C-nucleoside Antibiotics. Cell Chem Biol 25:540-549.e4
Maffioli, Sonia I; Sosio, Margherita; Ebright, Richard H et al. (2018) Discovery, properties, and biosynthesis of pseudouridimycin, an antibacterial nucleoside-analog inhibitor of bacterial RNA polymerase. J Ind Microbiol Biotechnol :
Walker, Scott S; Degen, David; Nickbarg, Elliott et al. (2017) Affinity Selection-Mass Spectrometry Identifies a Novel Antibacterial RNA Polymerase Inhibitor. ACS Chem Biol 12:1346-1352
Maffioli, Sonia I; Zhang, Yu; Degen, David et al. (2017) Antibacterial Nucleoside-Analog Inhibitor of Bacterial RNA Polymerase. Cell 169:1240-1248.e23
Lin, Wei; Mandal, Soma; Degen, David et al. (2017) Structural Basis of Mycobacterium tuberculosis Transcription and Transcription Inhibition. Mol Cell 66:169-179.e8
Yu, Libing; Winkelman, Jared T; Pukhrambam, Chirangini et al. (2017) The mechanism of variability in transcription start site selection. Elife 6:

Showing the most recent 10 out of 32 publications