The structure of premature core enzyme, an obligatory intermediate in both in vivo and in vitro assembly of Escherichia coli DNA-dependent RNA polymerase, was compared with that of native core enzyme. Though this assembled but inactive form of core enzyme harbors the gross conformation similar to that of native enzyme, minor and presumably local differences exist, which were identified by near-ultraviolet circular dichroism spectra, tritium-hydrogen exchange rate, protease sensitivity, intersubunit cross-linking rate by bifunctional reagents, sedimentation behavior, and elution profile from phosphocellulose. Taken together these results indicate that the core enzyme subunits are loosely associated in the premature core. The temperature-dependent maturation is required for the core subunits to be tightly associated, leading to the formation of structurally stable and functionally active RNA polymerase.