The Core Subunit Structure in RNA Polymerase Holoenzyme Determined by Neutron Small‐Angle Scattering

Abstract

The core subunit arrangement α2‐β‐β′ within DNA‐dependent RNA polymerase holoenzyme α2ββ′σ from Escherichia coli was investigated by neutron small‐angle scattering using label triangulation. The quaternary structure of multisubunit biomolecules can be studied by this new method if total reconstitution works in a quantitative way and if extensive replacement of C‐bound hydrogen (H) by deuterium (2H) is possible. A substitution of the selected subunits by their fully deuterated analogues was used for the analysis of the overall shapes of the core subunits α2, β and β′in situ and for the determination of the intersubunit centre‐to‐centre distances. The contrast between the buffer and the remaining ‘hydrogenated’ enzyme vanishes if the buffer contains 42%2H2O (matching of scattering length densities). The isotopic hybridization of the enzyme fulfils the conditions of isomorphous replacement as required: molecular functions, like enzyme activity, were completely preserved. The orientations of the core subunits within the holoenzyme were derived by comparing theoretical and experimental pair distance distribution functions, P(r), obtained from the scattering in tensity differences of the pair‐labelled (e.g. both β and β′ labelled) and both mono‐labelled molecules by direct Fourier transformations. Additional, the subunit shapes were refined by P(r) analyses. The arrangement of the stable core structure within the holoenzyme, which contains σ as a dissociable factor, is presented in a three‐dimensional model. Copyright © 1980, Wiley Blackwell. All rights reserved