The long-term objectives of this research are a) to determine the general biophysical principles involved in specificity and stability of protein-DNA complexes, and b) to apply these principles to understand the thermodynamic and kinetic basis of regulation of transcription initiation at prokaryotic promoters. Our fundamental biophysical strategy is to use ions and other physical variables to investigate the effects of changes in DNA recognition sequence on the thermodynamics and mechanisms of site- specific protein-DNA interactions, as well as to investigate important facilitating or competing multiple equilibria in these systems. Our major specific aims are 1) Kinetics and Mechanisms of RNA Polmerase-Promoter Interactions. Fluorescence-detected abortive initiation (FDAI) and filter-binding experiments on the strong lambda PR promoter and the weak lambda PRMupl promoter are proposed to characterize the intermediates on the pathway to open- complex formation and to determine the mechanistic steps affected by physical and chemical variables (supercoiling, temperature, pH, salt, effectors). Experiments on a family of promoter sequences spanning the range between the weak promoter lambda PRMupl and the strong consensus promoter sequence are proposed, in order to relate changes in DNA sequence and functional groups to individual mecha- nistic steps. 2) Thermodvnamics of lac Repressor-lac Operator Interactions. The thermodynamic origins of specificity and stability of binding of lac repressor to natural {Oc} and synthetic variants of the lac operator site are being examined by filter binding. Questions that will be addressed include our hypothesis of adaptability in recognition, the origin of the entropic driving force, and the question of whether noncovalent contacts provide independent (additive) or context-dependent non-additive) contributions to the binding free energy. This research will lead to a more detailed understanding of the relationship between structure {DNA sequence, protein conformation and composition} and function of two now-classical gene regulatory systems in vitro. Both the thermodynamic and mechanistic questions that we pose and the strategies (e.g. use of physical variables, expecially T, pH, ion concentrations) that we use to answer them are of general importance and applicability, so this work will serve as a model for studies on other systems. Since these regulatory systems can be studied at a quantitative level in vivo, our in vitro conclusions can be tested for physiological relevance.
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