Transcription initiation by DNA-dependent RNA polymerases (RNAP) at promoter DNA sequences involves large-scale conformational changes driven by binding free energy. The long term goal of this research on the representative E, coli enzyme is to characterize the sequence of these conformational changes and to determine their roles in creating the transcription bubble and active site for RNA synthesis. In the next project period, kinetic studies of association and dissociation will be performed in concert with low resolution structural characterizations (chemical and enzymatic footprinting;fluorescence resonance energy transfer (FRET)) to determine what conformational changes occur in each step, and how each sets the stage for the next.
Key specific aims i nclude elucidating how wrapping of nonspecific DNA sequences 60-80 base pairs upstream from the start site greatly accelerates open complex formation, and identifying when rearrangements in key regions of RNA polymerase (e.g. ejection of sigma70 masking domain from the active site channel, folding of unstructured regions of polymerase) occur. To this end, open complex formation by wild type and deletion polymerase mutants at promoter variants that delete upstream or downstream sequences will be characterized. Significant but simpler systems exhibiting analogous large-scale conformational changes will be studied in parallel: specific binding of 2 transcription factors (lac repressor dimer headpiece, IHF);and nonspecific binding of 2 nucleoid proteins (HU, IHF). Effects of temperature and solution variables on the thermodynamics of formation of a wrapped IHF-H'DNA interface will be measured by isothermal titration calorimetry (ITC) and FRET to obtain and interpret the thermodynamic signatures of DNA wrapping. Roles of multiple binding modes and cooperativity in nonspecific binding of HU will be determined using ITC. Coupling of local folding and formation of protein interfaces to operator binding will be investigated for the crosslinked dimeric headpiece by ITC, circular dichroism and a competitive nitrocellulose filter assay with intact lac repressor tetramer. Advances in the understanding of the mechanism of transcription initiation impacts 2 main areas of human health. First, understanding how transcriptional networks respond to developmental and environmental cues is fundamental to identifying and addressing failures in regulation that cause human diseases such as cancer. Secondly, human illnesses caused by bacterial infection (e.g. tuberculosis) wreak havoc on public health. Results generated by this proposal will define bacteria-specific aspects of initiation, and thus, reveal novel targets for the next generation of antibiotics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM023467-34
Application #
7753156
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Preusch, Peter C
Project Start
1977-01-01
Project End
2012-08-31
Budget Start
2010-01-01
Budget End
2012-08-31
Support Year
34
Fiscal Year
2010
Total Cost
$483,778
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Drennan, Amanda; Kraemer, Mark; Capp, Michael et al. (2012) Key roles of the downstream mobile jaw of Escherichia coli RNA polymerase in transcription initiation. Biochemistry 51:9447-59
Saecker, Ruth M; Record Jr, M Thomas; Dehaseth, Pieter L (2011) Mechanism of bacterial transcription initiation: RNA polymerase - promoter binding, isomerization to initiation-competent open complexes, and initiation of RNA synthesis. J Mol Biol 412:754-71
Koh, Junseock; Shkel, Irina; Saecker, Ruth M et al. (2011) Nonspecific DNA binding and bending by HUαβ: interfaces of the three binding modes characterized by salt-dependent thermodynamics. J Mol Biol 410:241-67
Gries, Theodore J; Kontur, Wayne S; Capp, Michael W et al. (2010) One-step DNA melting in the RNA polymerase cleft opens the initiation bubble to form an unstable open complex. Proc Natl Acad Sci U S A 107:10418-23
Kontur, Wayne S; Capp, Michael W; Gries, Theodore J et al. (2010) Probing DNA binding, DNA opening, and assembly of a downstream clamp/jaw in Escherichia coli RNA polymerase-lambdaP(R) promoter complexes using salt and the physiological anion glutamate. Biochemistry 49:4361-73
Capp, Michael W; Pegram, Laurel M; Saecker, Ruth M et al. (2009) Interactions of the osmolyte glycine betaine with molecular surfaces in water: thermodynamics, structural interpretation, and prediction of m-values. Biochemistry 48:10372-9
Schroeder, Lisa A; Gries, Theodore J; Saecker, Ruth M et al. (2009) Evidence for a tyrosine-adenine stacking interaction and for a short-lived open intermediate subsequent to initial binding of Escherichia coli RNA polymerase to promoter DNA. J Mol Biol 385:339-49
Pegram, Laurel M; Record Jr, M Thomas (2009) Quantifying the roles of water and solutes (denaturants, osmolytes, and Hofmeister salts) in protein and model processes using the solute partitioning model. Methods Mol Biol 490:179-93
Vander Meulen, Kirk A; Davis, Jared H; Foster, Trenton R et al. (2008) Thermodynamics and folding pathway of tetraloop receptor-mediated RNA helical packing. J Mol Biol 384:702-17
Koh, Junseock; Saecker, Ruth M; Record Jr, M Thomas (2008) DNA binding mode transitions of Escherichia coli HU(alphabeta): evidence for formation of a bent DNA--protein complex on intact, linear duplex DNA. J Mol Biol 383:324-46

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