Nonspecific Interactions of Escherichia coli RNA Polymerase with Native and Denatured DNA: Differences in the Binding Behavior of Core and Holoenzyme

Pieter L. deHaseth, Timothy M. Lohman, Richard R. Burgess, M. Thomas Record, Pieter L. deHaseth, Timothy M. Lohman

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We have investigated the nonspecific interactions of Escherichia coli RNA polymerase core and holoenzyme with double-stranded (ds) and single-stranded (ss) DNA. Binding constants for these interactions as functions of such solution variables as monovalent and/or divalent cation concentration, temperature, or pH were determined by the method of deHaseth et al. [deHaseth, P. L., Gross, C. A., Burgess, R. R. and Record, M. T. (1977), Biochemistry 16, 4777-4783] from analysis of the elution of the proteins from small columns containing immobilized DNA. This technique, although as yet empirical, has been demonstrated to yield accurate binding constants for the nonspecific interaction of lac repressor with ds DNA. We find that observed binding constants (Kobsd) are extraordinarily sensitive functions of the monovalent cation concentration for the interactions of both core and holoenzyme with ds DNA. In the absence of divalent cations, the derivatives -(d log Kobsd/d log [Na+]) are 11 ± 2 for the h lo-ds DNA interaction and 21 ± 3 for the core-ds DNA interaction. Consequently, approximately 11 and 21 low-molecular-weight ions are released, in the thermodynamic sense, in the formation of the holo-ds and core-ds complexes, respectively (Record, M. T., Jr., Lohman, T. M., and deHaseth, P. L. (1976), J. Mol. Biol. 107, 145-158; Record, M. T., Jr., Anderson, C. F., and Lohman, T. M. (1978), Q. Rev. Biophys., in press). Ion release is a thermodynamic driving force for these nonspecific interactions and causes the stability of the complexes to increase very substantially with a reduction in monovalent ion concentration. Possible molecular models which account for the different salt sensitivities of the holo-ds and core-ds complexes are discussed. Effects of the competitive ligand Mg2+ on these interactions are also examined. Substantial ion release (~18 monovalent ions) also accompanies the interaction of either holo or core polymerase with ss DNA. Over the range of ion concentrations investigated, the holo-ss interaction is substantially stronger than the core-ss interaction; furthermore, we conclude that the interactions of polymerase with ss DNA are, in general, stronger than the nonspecific interactions of the enzyme with ds DNA. It is likely that the nonspecific interactions of RNA polymerase with DNA have physiological relevance. Not only is it plausible to assume that the same regions of the protein are involved in both specific and nonspecific interactions, but in addition nonspecific interactions of RNA polymerase and DNA may play a role in determining the availability of this protein, in both the thermodynamic and the kinetic sense, for promoter binding and RNA chain initiation [von Hippel, P. H., Revzin, A., Gross, C. A., and Wang, A. C. (1974), Proc. Natl. Acad. Sci. U.S.A. 71, 4808-4812], Consequently, the strong dependences of the nonspecific interactions of RNA polymerase on ionic conditions suggest the possibility of a modulating role of ion concentrations in the control of transcription.

Original languageEnglish
Pages (from-to)1612-1622
Number of pages11
Issue number9
StatePublished - Jan 1 1978


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