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Regulatory factors acting at the bacteriophage Mu middle promoter
1Department of Microbiology and Immunology, University of Tennessee-Memphis 38163, USA.
Abstract:
Lytic development of bacteriophage Mu proceeds through three phases of transcription: early, middle, and late. Initiation of middle transcription from Pm requires the phage-encoded activator, Mor. An examination of the sequences surrounding the promoter revealed possible binding sites for Mu proteins A and c, as well as for Escherichia coli integration host factor. Promoter fragments containing 5' and 3' deletions were fused to the lacZ reporter gene and assayed for activity after induction of a Mu prophage or a plasmid-borne mor gene. Sequences upstream of position -62 and downstream of +10 were dispensable for promoter activity. In DNase I footprinting with both crude extract and purified protein, Mor protected Pm sequences from position -56 to -33. Mutations disrupting the dyad symmetry of the terminator of early transcription overlapping the Mor binding site did not reduce promoter activity, suggesting that the symmetry per se is not required for Mor binding or Pm activation. Purified Mu lysogenic repressor (c) also bound to Pm, overlapping the Mor binding site. Production of large amounts of repressor in vivo reduced Mor-dependent promoter activity nearly 10-fold. Promoters with mutations in the repressor binding site showed a reduction in this repressor-mediated inhibition of Pm activity.
Insights
Bacteriophage Mu middle transcription initiation requires the Mor activator. The Mor protein binds to the Pm promoter, and its activity is regulated by the Mu repressor protein c.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Bacteriophage Mu lytic development involves sequential transcriptional phases: early, middle, and late.
- Initiation of middle transcription from the Pm promoter is dependent on the phage-encoded activator, Mor.
- The Pm promoter region contains potential binding sites for Mu proteins A and c, and E. coli integration host factor.
Purpose of the Study:
- To investigate the regulatory elements and protein interactions involved in bacteriophage Mu middle transcription initiation.
- To identify the specific binding site of the Mor activator on the Pm promoter.
- To elucidate the role of the Mu repressor (c) in modulating Pm promoter activity.
Main Methods:
- Deletion analysis of promoter fragments fused to the lacZ reporter gene.
- DNase I footprinting assays with crude extracts and purified Mor protein.
- Site-directed mutagenesis of the Pm promoter and repressor binding site.
Main Results:
- Sequences between -62 and +10 of the Pm promoter are essential for activity.
- Mor protein protects the Pm promoter region from -56 to -33.
- The dyad symmetry of the early transcription terminator overlapping the Mor binding site is not required for Mor binding or Pm activation.
- Mu repressor (c) binds to the Pm promoter, overlapping the Mor binding site, and inhibits Mor-dependent activity.
- Mutations in the repressor binding site reduce the inhibitory effect of the repressor.
Conclusions:
- The Mor activator binds to a specific site on the Pm promoter, regulating middle transcription.
- The Mu repressor (c) antagonizes Mor activity by binding to the same promoter region.
- These findings provide insights into the complex transcriptional regulation during bacteriophage Mu lytic development.