Transcription initiation by bacterial RNA polymerase (RNAP) involves a series of steps: (i) RNAP binds to promoter DNA, yielding an RNAP-promoter closed complex; (ii) RNAP clamps tightly onto promoter DNA, yielding an RNAP-promoter intermediate complex; (iii) RNAP melts equal to approximately 14 nucleotides of promoter DNA, yielding a catalytically competent RNAP-promoter open complex; (iv) RNAP initiates synthesis of RNA, yielding an RNAP-promoter initial transcribing complex; and (v) RNAP breaks its interactions with promoter DNA--""""""""escapes""""""""--yielding an RNAP-DNA elongation complex. Each of these steps is a potential target for transcriptional regulators. Understanding transcription initiation and transcriptional regulation will require defining the structure of the RNAP-promoter complex at each step defining the structural transitions at each step, defining kinetics of structural transitions, and defining mechanisms by which regulators affect structural transitions. The proposed work will use fluorescence resonance energy transfer (FRET), single-molecule FRET, stopped-flow FRET, and kinetic photocrosslinking to address four specific aims:
Specific Aim 1 : To analyze the structure of RNAP holoenzyme.
Specific Aim 2 : To analyze the structures of trapped RNAP-promoter complexes.
Specific Aim 3 : To analyze the mechanism of entry of RNAP into promoter DNA.
Specific Aim 4 : To analyze the mechanism of escape of RNAP from promoter DNA. The results will contribute to understanding bacterial transcription initiation, to understanding bacterial transcriptional regulation, and to design and synthesis of low-molecular-weight inhibitors of bacterial transcription, for application in antimicrobial therapy. Since eukaryotic RNAP subunits show sequence, structural, and mechanistic similarities to bacterial RNAP subunits, the results also will contribute to understanding eukaryotic transcription initiation and regulation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041376-14
Application #
6476499
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Chin, Jean
Project Start
1988-12-01
Project End
2004-11-30
Budget Start
2001-12-01
Budget End
2002-11-30
Support Year
14
Fiscal Year
2002
Total Cost
$263,689
Indirect Cost
Name
Rutgers University
Department
Type
Organized Research Units
DUNS #
038633251
City
New Brunswick
State
NJ
Country
United States
Zip Code
08901
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
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:
Bird, Jeremy G; Nickels, Bryce E; Ebright, Richard H (2017) RNA Capping by Transcription Initiation with Non-canonical Initiating Nucleotides (NCINs): Determination of Relative Efficiencies of Transcription Initiation with NCINs and NTPs. Bio Protoc 7:

Showing the most recent 10 out of 39 publications