Activation of the bacteriophage Mu lys promoter by Mu C protein requires the sigma 70 subunit of Escherichia coli RNA polymerase

Abstract

Bacteriophage Mu C protein, a product of the middle operon, is required for activation of the four Mu late promoters. To address its mechanism of action, we overproduced the approximately 16.5-kilodalton C protein from a plasmid containing the C gene under the control of a phage T7 promoter and ribosome-binding site. A protein fraction highly enriched for Escherichia coli RNA polymerase (E sigma 70) and made from the overproducing strain was able to activate transcription in vitro from both the tac promoter (Ptac) and a Mu late promoter, Plys. The behavior of Plys was similar in vivo and in vitro; under both conditions, transcription was C dependent and the RNA 5' ends were identical. When anti-sigma 70 antibody was added to C-dependent transcription reactions containing both Ptac and Plys templates, transcription from both promoters was inhibited; transcription was restored by the addition of excess E sigma 70. This result suggests that C-dependent activation of Plys requires sigma 70. Further supporting evidence was provided by a reconstitution experiment in which an E sigma 70-depleted fraction containing C was unable to activate transcription from Plys unless both purified sigma 70 and core polymerase were added. These results strongly suggest that C is not a new sigma factor but acts as an activator for E sigma 70-dependent transcription.