Escherichia coli RNA polymerase subunit ω and its N-terminal domain bind full-length β′ to facilitate incorporation into the α2β subassembly

Abstract

The ω subunit of Escherichia coli RNA polymerase, consisting of 90 amino acids, is present in stoichiometric amounts per molecule of core RNA polymerase (α2ββ′). The presence of ω is necessary to restore denatured RNA polymerase in vitro to its fully functional form, and, in an ω-less strain of E. coli, GroEL appears to substitute for ω in the maturation of RNA polymerase. The X-ray structure of Thermus aquaticus core RNA polymerase suggests that two regions of ω latch on to β′ at its N-terminus and C-terminus. We show here that ω binds only the intact β′ subunit and not the β′ N-terminal domain or β′ C-terminal domain, implying that ω binding requires both these regions of β′. We further show that ω can prevent the aggregation of β′ during its renaturation in vitro and that a V8-protease-resistant 52-amino-acid-long N-terminal domain of ω is sufficient for binding and renaturation of β′. CD and functional assays show that this N-terminal fragment retains the structure of native ω and is able to enhance the reconstitution of core RNA polymerase. Reconstitution of core RNA polymerase from its individual subunits proceeds according to the steps α + α → α2 + β → α2β + β′ → α2ββ′. It is shown here that ω participates during the last stage of enzyme assembly when β′ associates with the α2β subassembly.