Subunit topography of RNA polymerase from Escherichia coli. A cross-linking study with bifunctional reagents

Abstract

The quaternary structures of E. coli DNA-dependent RNA polymerase holoenzyme (.alpha.2.beta..beta.''.sigma.) and core enzyme (.alpha.2.beta..beta.'') were 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 .beta. and .beta.'' subunits constitute the backbone of both core and holoenzyme, while .sigma. and 2 .alpha. subunits interact with this structure along the contact domain of .beta. and .beta.'' subunits. In holoenzyme, .sigma. subunit is in the vicinity of at least one .alpha. subunit. The 2 .alpha. subunits are close to each other in holoenzyme, core enzyme and the isolated .alpha.2.beta. 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 MW lower than that of native core and holoenzyme, respectively. The structural information obtained for RNA polymerase and its subcomplexes has important implications for the enzyme-promoter recognition and the mechanism of subunit assembly of the enzyme.