We used binding assays and other approaches to identify fragments of the Escherichia coli RNAP beta subunit involved in the obligatory interaction with the alpha subunit to form the stable assembly intermediate alpha2beta as well as in the interaction to recruit the beta' subunit into the alpha2beta sub-assembly. We show that two regions of evolutionarily conserved sequence near the C terminus of beta (conserved regions H and I) are central to the assembly of RNAP and likely make subunit-subunit contacts with both alpha and beta'.