The beta ' subunit of Escherichia coli RNA polymerase is not required for interaction with initiating nucleotide but is necessary for interaction with rifampicin.

Using a modification of a highly selective affinity labeling protocol, we demonstrated that the alpha(2)beta subassembly of Escherichia coli RNA polymerase efficiently and specifically interacts with the initiating purine nucleotide. Isolated beta is also active in this reaction. In contrast, neither beta nor alpha(2)beta 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, beta'. However, the rifampicin binding center is formed only in the alpha(2)betabeta' core enzyme. We interpret our results in light of the high resolution structure of core RNA polymerase from Thermus aquaticus.