Bacteriophage Mu Mor Protein Requires Sigma 70 to Activate the Mu Middle Promoter

Transcription during the bacteriophage Mu lytic cycle occurs in three phases: early, middle, and late. Middle transcription requires the early gene product Mor for its activation. Mor protein overproduction was accomplished by fusing the mor gene to an efficient phage T7 promoter and translation initiation region. A protein fraction highly enriched for Escherichia coli RNA polymerase (E sigma 70) from the Mor-overproducing strain was able to activate transcription from both the tac promoter (Ptac) and the Mu middle promoter (Pm) in vitro. Transcription initiation from Pm was Mor dependent, and the RNA 5' end was identical to that of in vivo RNA. Addition of anti-sigma 70 antibody to transcription reactions containing Ptac and Pm resulted in inhibition of transcription from both promoters; addition of purified sigma 70 restored transcription. These results indicate that Mor-dependent activation requires sigma 70 and therefore imply that Mor is not an alternate sigma factor. This conclusion was further substantiated by a reconstitution experiment with purified proteins in which all three components, Mor, sigma 70, and core RNA polymerase, were required for Pm-dependent transcription in vitro. The sigma 70 dependence of Mor-specific transcription and the amino acid sequence similarity between Mor and C (an activator for Mu late transcription) both support the hypothesis that Mor functions mechanistically as an activator protein.