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

The core subunit arrangement α 2 ‐β‐β′ within DNA‐dependent RNA polymerase holoenzyme α 2 ββ′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 ( 2 H) 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 α 2 , 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% 2 H 2 O (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.