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Bacteriophage Mu Mor protein requires sigma 70 to activate the Mu middle promoter
1Department of Microbiology and Immunology, University of Tennessee-Memphis 38163.
Abstract:
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.
Insights
The bacteriophage Mu Mor protein activates middle-phase transcription by interacting with sigma 70 (σ70) and RNA polymerase, not by acting as an alternative sigma factor. This clarifies Mor
Area of Science:
- Molecular Biology
- Bacteriophage Genetics
- Gene Regulation
Background:
- Bacteriophage Mu lytic cycle involves sequential early, middle, and late transcription phases.
- Middle transcription activation is dependent on the early gene product, Mor.
- Understanding Mor's mechanism is key to deciphering phage transcription regulation.
Purpose of the Study:
- To investigate the role of the Mor protein in bacteriophage Mu middle transcription activation.
- To determine if Mor functions as an alternative sigma factor or an activator protein.
- To elucidate the molecular mechanism of Mor-mediated transcriptional activation.
Main Methods:
- Overproduction of Mor protein by fusing its gene to a T7 promoter.
- In vitro transcription assays using Escherichia coli RNA polymerase (Eσ70) and purified components.
- Utilizing anti-sigma 70 antibodies and purified sigma 70 to probe protein interactions.
- Reconstitution experiments with purified Mor, sigma 70, and core RNA polymerase.
Main Results:
- Mor protein overproduction enabled in vitro transcription activation from the Mu middle promoter (Pm).
- Mor-dependent transcription from Pm required sigma 70, as shown by antibody inhibition and restoration with purified sigma 70.
- Reconstitution experiments confirmed that Mor, sigma 70, and core RNA polymerase are all essential for Pm-dependent transcription.
- Mor does not function as an alternative sigma factor but as an activator protein.
Conclusions:
- Mor protein acts as a transcriptional activator, requiring sigma 70 for bacteriophage Mu middle transcription.
- The findings support a model where Mor interacts with the RNA polymerase holoenzyme to facilitate transcription initiation.
- Mor's mechanism is distinct from that of alternative sigma factors, highlighting diverse regulatory strategies in phage biology.