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Mutational analysis of a C-dependent late promoter of bacteriophage Mu
1Department of Microbiology and Immunology, University of Tennessee, Memphis 38163.
Abstract:
Late transcription of bacteriophage Mu initiates at four promoters, P(lys), PI, PP and Pmom, and requires the Mu C protein and the host RNA polymerase. Promoter-containing DNA fragments extending approximately 200 bp upstream and downstream of the 5' starts of the lys, I and P transcripts were cloned into a multicopy lacZ-expression plasmid. Promoter activity, assayed by beta-galactosidase expression, was determined under two different conditions: (1) with C provided from a compatible plasmid in the absence of other Mu factors and (2) with C provided from an induced Mu prophage. beta-galactosidase activities were greatest for P(lys), intermediate for PI, and lowest for PP. Similar analysis of plasmids containing nested sets of deletions removing 5' or 3' sequences of P(lys) demonstrated that a 68-bp region was sufficient for full activity. Point mutations were generated within the 68-bp region by mutagenic oligonucleotide-directed PCR (Mod-PCR). Properties of the lys promoter mutants indicated that, in addition to the -10 region, a 19-bp region from -52 to -34 containing the C footprint is required for C-dependent promoter activity.
Insights
Bacteriophage Mu late transcription relies on the Mu C protein and host RNA polymerase. A specific 19-bp region within the P(lys) promoter is crucial for this C-dependent activity.
Area of Science:
- Molecular Biology
- Microbiology
- Virology
Background:
- Bacteriophage Mu employs distinct promoters for late gene transcription.
- The Mu C protein and host RNA polymerase are essential for initiating late transcription.
Purpose of the Study:
- To characterize the regulatory elements of bacteriophage Mu late transcription promoters.
- To identify the specific DNA sequences and protein interactions required for promoter activity.
Main Methods:
- Cloning of promoter-containing DNA fragments into a lacZ-expression plasmid.
- Assaying promoter activity via beta-galactosidase expression under varying conditions.
- Deletion analysis and site-directed mutagenesis (Mod-PCR) of the P(lys) promoter.
Main Results:
- Promoter activity varied, with P(lys) > PI > PP.
- A 68-bp region of P(lys) was sufficient for full promoter activity.
- A 19-bp region (-52 to -34) within P(lys), including the C footprint, is essential for Mu C protein-dependent transcription.
Conclusions:
- The P(lys) promoter exhibits the highest activity among the characterized late promoters.
- Specific DNA sequences and the Mu C protein are critical for regulating bacteriophage Mu late transcription initiation.