Our long term goals are to establish general thermodynamic and kinetic- mechanistic principles which govern macromolecular recognition and protein- nucleic acid interactions (PNAI) in aqueous solution, and to apply these in relating structure to function. We propose to obtain a quantitative understanding of function of key site-specific PNAI involved in regulation of transcription initiation in E. coli (Lac repressor-lac operator, RNA polymerase-promoter), which will extend to other pro- and eukaryotic gene regulatory proteins which control the processes of normal and abnormal cell development. A) The thermodynamic signatures of coupled conformational changes in DNA (e.g. kinking, smooth bending, melting) and in the protein (e.g. folding, hinge-bending) in PNAI will be deduced and used to interpret the thermodynamics (delta-C-obs, TS, TH, SK-obs) of interactions involving conformational changes. B) Contributions of the Lacl headpiece (HP) and the core of the Lacl tetramer to operator binding will be dissected. Values of delta-C-obs, TS, TH and SK-obs will be determined for interactions of wildtype and variant Lacl HP with operator by calorimetry and sedimentation equilibrium to test our hypothesis that HP is partially unstructured in the absence of DNA, and folds to a unique structure upon binding. Tetramer-operator equilibria will be investigated by filter binding as a function of length of flanking DNA to test our proposals that coulombic interactions between the Lacl core and flanking nonoperator DNA (via wrapping and looping) stabilize 1:1 complexes, and competitively destabilize the 2:1 complex at low salt. C) The mechanistic pathway, intermediates, and bottleneck kinetic step in isomerization of the """"""""closed"""""""" complex to the functional """"""""open"""""""" complex at the APR promoter will be determined using RNA polymerases from E. coli (E- sigma70) and a thermophilic eubacterium. Thermodynamic, kinetic and footprinting studies will be used to test our proposal that closing the jaws of polymerase and the initial stages of opening the promoter occur together and are the kinetic bottleneck. Intermediate open-promoter complexes will be characterized to study the roles of Mg2+ and the sequence of steps in opening the transcription start site. Fluorescence studies of the thermodynamics and kinetics of binding sigma(70) to core polymerase will test the proposals that binding of sigma(70) opens the jaws of core and unmasks the DNA recognition domain of sigma(70).

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 #
5R37GM023467-24
Application #
6138365
Study Section
Biochemistry Study Section (BIO)
Program Officer
Lewis, Catherine D
Project Start
1977-01-01
Project End
2001-12-31
Budget Start
2000-01-01
Budget End
2000-12-31
Support Year
24
Fiscal Year
2000
Total Cost
$347,374
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 HU??: 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; Saecker, Ruth M; Record Jr, M Thomas (2008) Formation of a wrapped DNA-protein interface: experimental characterization and analysis of the large contributions of ions and water to the thermodynamics of binding IHF to H'DNA. J Mol Biol 377:9-27
Kontur, Wayne S; Saecker, Ruth M; Capp, Michael W et al. (2008) Late steps in the formation of E. coli RNA polymerase-lambda P R promoter open complexes: characterization of conformational changes by rapid [perturbant] upshift experiments. J Mol Biol 376:1034-47

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