Subunit Topography of RNA Polymerase from Escherichia coli. A Cross-Linking Study with Bifunctional Reagents

The quaternary structures of Escherichia coli DNA-dependent RNA polymerase holoenzyme (α₂ββ′σ) and core enzyme (α₂ββ′) have been investigated by chemical cross-linking with a cleavable bifunctional reagent, methyl 4-mercaptobutyrimidate, and noncleavable reagents, dimethyl suberimidate and N,N′-(1,4-phenylene)bismaleimide. A model of the subunit organization deduced from cross-linked subunit neighbors identified by dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the large β and β′ subunits constitute the backbone of both core and holoenzyme, while σ and two α subunits interact with this structure along the contact domain of β and β′ subunits. In holoenzyme, σ subunit is in the vicinity of at least one a subunit. The two α subunits are close to each other in holoenzyme, core enzyme, and the isolated α₂β complex. Cross-linking of the “premature” core and holoenzyme intermediates in the in vitro reconstitution of active enzyme from isolated subunits suggests that these species are composed of subunit complexes of molecular weight lower than that of native core and holoenzyme, respectively. The structural information obtained for RNA polymerase and its subcomplexes has important implications for the enzymepromoter recognition as well as the mechanism of subunit assembly of the enzyme.