9] Reconstitution of RNA polymerase

Abstract

Publisher Summary The DNA-dependent RNA polymerase of Escherichia coli is composed of core enzyme and one of at least six molecular species of the σ subunit. Despite the complexity of its subunit composition and its extraordinarily large molecular size, RNA polymerase is one of the few large molecular assemblies that can be successfully reconstituted from isolated individual subunits. Core enzyme can be reconstituted by mixing α, β, and β' subunits under denaturing conditions, and then removing the denaturant by dialysis against the appropriate reconsitution buffer. The core enzyme reconstituted at low temperature is known to exist as an inactive premature from. It can be activated by heating at 30 ° for 30–60 min. Although the activated core enzyme can be used directly for many purposes, it contains considerable amounts of free subunits and α 2 β subassembly. For quantitative experiments, therefore, further purification of the reconstituted enzyme is essential.