Quantitative analysis of multiple-hit footprinting studies to characterize DNA conformational changes in protein-DNA complexes: Application to DNA opening by Eσ⁷⁰ RNA polymerase

Formation of many site-specific protein-nucleic acid complexes involves sequential conformational changes subsequent to initial binding which create functionally active assemblies. Characterization of population distributions and structural characteristics of intermediate and product conformations is necessary to understand both the mechanisms and the thermodynamics of these processes. For these purposes, here we develop the quantitative method of multiple hit footprinting (MHF), where chemical or enzymatic probing is performed as a function of either concentrations of the footprinting agent and/or time of exposure to it, in the multiple hit regime where many of the population or subpopulation of reactive DNA molecules are modified at more than one site. Properly controlled MHF experiments yield both the population distribution of different conformers and reactivity rate constants of the footprinting agent at all reactive positions in each conformer, which may be interpreted in terms of the accessibility of the site or the local concentration of the reagent. MHF experiments are particularly well-suited for dissecting effects at sites where unbound DNA is non-reactive and bound DNA is reactive with base-specific probes (e.g. KMnO₄, DMS). We suggest that this method will also be applicable to analysis of enhancements in reactivity of other footprinting agents (e.g. DNase I, HO·). To demonstrate the utility of the MHF analysis, we quantify fragment distributions and individual site reactivities from multiple-hit KMnO, footprinting of the non-template strand of Eσ⁷⁰ RNA polymerase-λP(R) promoter DNA complexes populated at binding equilibrium at 37°C and transiently populated at a fixed time after a temperature downshift from 37°C to 0°C. For this system, a MHF analysis directly addresses the following questions: (i) what fraction of the population of promoter DNA molecules is open in the vicinity of the transcription start site (RP(o)) both at 37°C and (transiently) after a downshift to 0°C; (ii) does opening of the start site region in RP(o) occur entirely in one mechanistic step at the λP(R), promoter and (iii) does the structure of RP(o) vary with temperature? In addition, we use the MHF-determined population distribution of KMnO4-reactive (RP(o)) and non-reactive promoter DNA to normalize the biphasic kinetics of decay of RP(o) to free promoter DNA after a 37°to 0°C temperature downshift, and thereby characterize the kinetics of the conformational changes involved in forming RP(o).