Self-Association of Escherichia coli DNA-Dependent RNA Polymerase Core Enzyme

Simon J. Harris, Robley C. Williams, J. Ching Lee

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

The extent of self-association of Escherichia coli DNA-dependent RNA polymerase core enzyme has been investigated by velocity sedimentation as a function of both NaCl and protein concentrations. The core enzyme, existing as essentially monomeric species having a sedimentation coefficient of 13.1 S at NaCl concentrations greater than 0.2 M, undergoes reversible self-association at lower salt concentrations. Estimates for the stoichiometry of association and equilibrium constants of reaction were determined from the effect of protein concentration on the weight-average sedimentation coefficient measured at different NaCl concentrations. Data analysis by a nonlinear curve-fitting procedure indicated that protein self-association is best described by a sequential model characterized by weaker association constants for each additional step of oligomerization, and any model that involves cooperative formation of oligomeric species can be excluded. These findings are at variance with the conclusion of a previous study [Shaner, S. L., Platt, D. M., Wensley, C. G., Yu, H., Burgess, R. R., & Record, M. T. (1982) Biochemistry 26, 5539-5551] which suggested that core RNA polymerase exists in equilibrium between monomeric and tetrameric forms of the enzyme and excluded the existence of intermediate species. Simulation of sedimentation velocity boundary and gradient profiles are used to assess the validity of both models of association of core protein. It was clear that had the core enzyme undergone a cooperative monomer ↔ tetramer mode of association, then bimodality would have been observed in the derivative tracings of the sedimentation boundary under these experimental conditions. Nevertheless, no such observation was reported by Shaner et al. and this study. The sequential model favored by the results of this study is consistent with the proposed model resulted from a small-angle X-ray study [Heumann, H., Meisenberger O., & Pilz, I. (1982) FEBS Lett. 138, 273-276], Further analysis of the data by the Wyman linkedfunction relationship [Wyman, J. (1964) Adv. Protein Chem. 19, 223-286] implies that core enzyme monomer loses approximately three counterions per contact upon association to higher oligomeric species.

Original languageEnglish (US)
Pages (from-to)8752-8762
Number of pages11
JournalBiochemistry
Volume34
Issue number27
DOIs
StatePublished - Jul 1995

ASJC Scopus subject areas

  • Biochemistry

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