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).
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