The β′ Subunit of Escherichia coli RNA Polymerase Is Not Required for Interaction with Initiating Nucleotide but Is Necessary for Interaction with Rifampicin

Abstract

Using a modification of a highly selective affinity labeling protocol, we demonstrated that the α2β subassembly of Escherichia coli RNA polymerase efficiently and specifically interacts with the initiating purine nucleotide. Isolated β is also active in this reaction. In contrast, neither β nor α2β is able to interact with a chimeric molecule composed of rifampicin attached to an initiation substrate. Based on these results, we conclude that the RNA polymerase initiation site, specific for purine nucleotides, which ultimately become the 5′-end of the transcript, is essentially complete in the absence of the largest subunit, β′. However, the rifampicin binding center is formed only in the α2ββ′ core enzyme. We interpret our results in light of the high resolution structure of core RNA polymerase from Thermus aquaticus.