The long term objectives of this research are to quantify contributions of noncovalent interactions to stability and specificity of protein-DNA complexes, and to develop principles of thermodynamic and mechanistic analyses of these interactions. In vivo, specificity in gene expression may be exhibited at the thermodynamic level (e.g. lac repressor, lac R) or the kinetic level (e.g. E sigma 70 RNA polymerase, RNAP). Relating sequence to function and understanding stability, specificity, and context-dependence of sequence variants in these systems require detailed thermodynamic and kinetic studies such as those proposed here. A) Key residues of lac R (e.g. tyr17) and of the symmetric lac operator O/sym (e.g. GTG at positions 4-6) identified by in vivo studies of variants, will be investigated in vitro. Thermodynamic effects of replacement of tyr17 by a functional group deletion (phe17) or substitutions (his 17, gln17, glu17) will be determined by 32P-DNA- detected filter-binding (FB). The extent and thermodynamic origins of context-dependence in the binding of lac R to O/sym operator variants at positions 4-6 (GTG) will also be studied. The thermodynamic contributions of key functional groups (e.g. the methyl group of T at position 3) on O/sym will be probed with selected base analogs (e.g. deletion of the methyl group by replacing T by U). B) Kinetic and mechanistic studies with RNAP will be performed to determine the roles of the conserved recognition regions of the promoter DNA and to test the """"""""bipartite"""""""" model of promoter function. Kinetic consequences of selected series of base pair substitutions in the -10 and -35 regions and changes in the spacer region of a consensus promoter construct will be investigated by FB and the fluorescence- detected abortive initiation (FDAI) assay. FDAI will be used to examine effects of lac R on the mechanistic steps of open complex formation in constructs in which a lac operator overlaps the lacUV5 or lambda P/R promoter. The Mg2+-driven interconversion of open complexes at the lambda P/R promoter will be further characterized using FDAI detected dissociation kinetics and appropriate chemical probes. C) The proposed key role of the hydrophobic effect in site-specific protein-DNA interactions will be further examined. Structural and thermodynamic data (heat capacity changes) for in-DNA and protein- protein interactions will be obtained and analyzed. The thermodynamic consequences of burial of nonpolar surface in specific protein-DNA interactions will be investigated by comparing heat capacity changes for binding of EcoRI endonuclease and a deletion mutant missing the N- terminal domain, and for binding of two GCN4 peptides, one with and one without the leucine zipper region.
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