This proposal focusses on transcription initiation and elongation by bacterial RNA polymerase (RNAP). Transcription initiation and elongation involve a series of steps: (i) RNAP binds to promoter DNA, yielding an RNAP-promoter closed complex;(ii) RNAP unwinds promoter DNA, yielding an RNAP-promoter open complex;(iii) RNAP synthesizes the first ~8 -15 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) RNAP breaks its interactions with promoter DNA and 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. Each of these steps is a potential target for transcriptional regulators. Understanding transcription initiation, transcription elongation, and transcriptional regulation will require defining the structural transitions in protein and DNA at each step, defining kinetics of structural transitions, and defining mechanisms by which transcriptional regulators affect structural transitions. The proposed work will use ensemble and single-molecule fluorescence resonance energy transfer, single-molecule nanomanipulation, biochemical methods, and genetic methods to address five specific aims:
Specific Aim 1 : Determination of the mechanism of scrunching Specific Aim 2: Determination of the role of scrunching Specific Aim 3: Determination of the mechanism of RNAP active-center-cleft loading Specific Aim 4: Detection and analysis of RNAP active-center conformational cycling Specific Aim 5: Determination of the target and mechanism of transcriptional regulators ppGpp and DksA The results will contribute to understanding bacterial transcription and transcriptional regulation, and will contribute to design and synthesis of small-molecule inhibitors of bacterial transcription, for application in antibacterial therapy. Since bacterial RNAP subunits show sequence, structural, and mechanistic similarities to eukaryotic RNAP subunits, the results also will contribute to understanding eukaryotic transcription and transcriptional regulation.

Public Health Relevance

Bacterial RNA polymerase (RNAP) is a molecular machine that carries out reactions essential for bacterial gene expression and bacterial growth. An important class of broad-spectrum antibacterial therapeutic agents functions 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 broad-spectrum antibacterial agents that function by inhibiting RNAP.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041376-24
Application #
8204495
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Preusch, Peter C
Project Start
1988-12-01
Project End
2013-01-31
Budget Start
2011-12-01
Budget End
2013-01-31
Support Year
24
Fiscal Year
2012
Total Cost
$374,706
Indirect Cost
$128,700
Name
Rutgers University
Department
None
Type
Organized Research Units
DUNS #
001912864
City
New Brunswick
State
NJ
Country
United States
Zip Code
08901
Vorobiev, Sergey M; Gensler, Yocheved; Vahedian-Movahed, Hanif et al. (2014) Structure of the DNA-binding and RNA-polymerase-binding region of transcription antitermination factor ?Q. Structure 22:488-95
Vvedenskaya, Irina O; Vahedian-Movahed, Hanif; Bird, Jeremy G et al. (2014) Interactions between RNA polymerase and the "core recognition element" counteract pausing. Science 344:1285-9
Robb, Nicole C; Cordes, Thorben; Hwang, Ling Chin et al. (2013) The transcription bubble of the RNA polymerase-promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: implications for transcription start-site selection. J Mol Biol 425:875-85
Grohmann, Dina; Nagy, Julia; Chakraborty, Anirban et al. (2011) The initiation factor TFE and the elongation factor Spt4/5 compete for the RNAP clamp during transcription initiation and elongation. Mol Cell 43:263-74
Srivastava, Aashish; Talaue, Meliza; Liu, Shuang et al. (2011) New target for inhibition of bacterial RNA polymerase: 'switch region'. Curr Opin Microbiol 14:532-43
Chakraborty, Anirban; Wang, Dongye; Ebright, Yon W et al. (2010) Azide-specific labeling of biomolecules by Staudinger-Bertozzi ligation phosphine derivatives of fluorescent probes suitable for single-molecule fluorescence spectroscopy. Methods Enzymol 472:19-30
Hudson, Brian P; Quispe, Joel; Lara-Gonzalez, Samuel et al. (2009) Three-dimensional EM structure of an intact activator-dependent transcription initiation complex. Proc Natl Acad Sci U S A 106:19830-5
Ho, Mary X; Hudson, Brian P; Das, Kalyan et al. (2009) Structures of RNA polymerase-antibiotic complexes. Curr Opin Struct Biol 19:715-23
Naryshkin, Nikolai; Druzhinin, Sergei; Revyakin, Andrei et al. (2009) Static and kinetic site-specific protein-DNA photocrosslinking: analysis of bacterial transcription initiation complexes. Methods Mol Biol 543:403-37
Goldman, Seth R; Ebright, Richard H; Nickels, Bryce E (2009) Direct detection of abortive RNA transcripts in vivo. Science 324:927-8

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