The Subunit Positions within RNA Polymerase Holoenzyme Determined by Triangulation of Centre‐to‐Centre Distances

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Abstract
The complete ‘centre‐of‐subunit structure’ of the multisubunit enzyme DNA‐dependent RNA polymerase was determined by triangulation of the subunit positions using the intersubunit distances calculated from scattering difference measurements and from the corresponding radii of gyration R. In addition to the centre‐to‐centre distances d between the core subunits α2, β and β′ presented in the preceding paper, the values of d between initiation factor σ and α2 (8.4 ± 1.6 nm), β (4.4 ± 2.2 nm) and β′ (10.7 ± 1.5 nm) were derived from R of σ (4.1 ± 0.3 nm) in situ and of the pairs α2‐σ (6.1 ± 0.4 nm), β‐σ (5.6 ± 0.3 nm) and β‐σ (7.5 ± 0.4 nm) within the holoenzyme (α2ββ′σ). The structural parameters of the subunits within their molecular complex are accessible for neutron small‐angle scattering measurements using labelling of the different subunits (deuteration), total reconstitution of isotopic hybrids, scattering length density matching of ‘hydrogenated’ molecular parts and extended exposure times because of weak scattering effects. The overall shape of σ bound to core enzyme (α2ββ′) proved to be identical (within experimental resolution) with σ in the isolated state measured recently by X‐ray small‐angle scattering. The refined shape of isolated σ was reduced to an ellipsoid which was orientated with respect to the core structure (α2‐β‐β′) in a ‘space‐filling’ way around the position of the a centre obtained by triangulation. The complete subunit arrangement of holoenzyme is shown in a three‐dimensional model.