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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
4R37GM041376-30
Application #
9312918
Study Section
Special Emphasis Panel (NSS)
Program Officer
Preusch, Peter
Project Start
1988-12-01
Project End
2023-01-31
Budget Start
2018-02-01
Budget End
2019-01-31
Support Year
30
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Rutgers University
Department
Type
Organized Research Units
DUNS #
001912864
City
Piscataway
State
NJ
Country
United States
Zip Code
08854
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
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:
Bird, Jeremy G; Zhang, Yu; Tian, Yuan et al. (2016) The mechanism of RNA 5? capping with NAD+, NADH and desphospho-CoA. Nature 535:444-7
Vvedenskaya, Irina O; Vahedian-Movahed, Hanif; Zhang, Yuanchao et al. (2016) Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection. Proc Natl Acad Sci U S A 113:E2899-905
Feng, Yu; Zhang, Yu; Ebright, Richard H (2016) Structural basis of transcription activation. Science 352:1330-3
Winkelman, Jared T; Vvedenskaya, Irina O; Zhang, Yuanchao et al. (2016) Multiplexed protein-DNA cross-linking: Scrunching in transcription start site selection. Science 351:1090-3
Feng, Yu; Degen, David; Wang, Xinyue et al. (2015) Structural Basis of Transcription Inhibition by CBR Hydroxamidines and CBR Pyrazoles. Structure 23:1470-1481
Hassan, Hossam M; Degen, David; Jang, Kyoung Hwa et al. (2015) Salinamide F, new depsipeptide antibiotic and inhibitor of bacterial RNA polymerase from a marine-derived Streptomyces sp. J Antibiot (Tokyo) 68:206-9
Chakraborty, Anirban; Mazumder, Abhishek; Lin, Miaoxin et al. (2015) Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation. Methods Mol Biol 1276:101-31

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