Structural analysis of the Bacillus subtilis δ factor: A protein polyanion which displaces RNA from RNA polymerase

Abstract

RNA polymerase from Bacillus subtilis is a complex mixture comprising a common core (ββ'α2), the 20.4 kDa delta (δ) protein, and one of several sigma (σ) specificity factors. The δ protein, together with several truncated variants, has been overproduced and purified from Escherichia coli. It is highly acidic (pI = 3.6) and contains two distinct regions, a 13 kDa amino-terminal domain with fairly uniform charge distribution and a glutamate and aspartate residue-rich carboxyl-terminal region. The purified amino-terminal domain (δN) contains 32% alpha-helix and 16% beta-sheet, as judged by circular dichroism analysis. In contrast, an 8.5 kDa tryptic fragment containing the carboxyl-terminal region (δC) is largely unstructured and highly charged (net charge of -47). RNA polymerase purified from a B. subtilis mutant with an insertion in the δ gene (rpoE::cat) contains a truncated δ protein, indicating that the amino-terminal domain is stable in vivo and contains a core-binding function. Addition of δ, but not σ(A) or δN, displaces RNA bound to RNA polymerase in a binary complex. The ability of δ to displace RNA efficiently requires the activities of both the amino-terminal core-binding domain and the polyanionic carboxyl-terminal region. Although δC can also displace nucleic acids from RNA polymerase, this activity requires the addition of a large molar excess of protein and is relatively non specific in that both DNA and RNA are displaced. This suggests that the function of the amino-terminal domain is to bind and orient the carboxyl-terminal region on the surface of RNA polymerase. © 1995 Academic Press Limited.