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Identification and characterization of the terminators of the lys and P transcripts of bacteriophage Mu
1Department of Microbiology and Immunology, University of Tennessee, Memphis 38163.
Abstract:
Transcription during the lytic cycle of phage Mu occurs in three phases: early, middle, and late. Late transcription requires the Mu C protein and initiates at four promoters: Plys, PI, PP, and Pmom. Northern blot analysis of total RNA isolated 30 min after heat induction of Mu cts lysogens demonstrated that the full-length lys and P transcripts were approximately 7.6 and 6.3 kb long, respectively. The 3' ends of the lys and P transcripts were further localized by S1 nuclease mapping to intergenic regions between G and I and between U and U' in both the G(+) and G(-) orientations of the invertible G segment, respectively. As expected, when DNA fragments containing these termination regions were cloned into plasmids between Pgal and the galK gene, they showed efficient termination activity, even in a Rho-deficient background. Deletion analysis indicated that efficient termination required the presence of potential RNA stem-loop structures immediately preceding the RNA 3' ends. For the P transcript from phage with the G(-) orientation, full termination activity required both the region containing the stem-loop structure and upstream sequences. Taken together, these results suggest that the transcription termination sites of the lys and P transcripts are Rho-independent terminators.
Insights
Late transcription in phage Mu requires the C protein and initiates at specific promoters. Termination sites for lys and P transcripts are identified as Rho-independent, relying on RNA stem-loop structures.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Bacteriophage Mu exhibits distinct transcriptional phases during its lytic cycle.
- Late transcription is regulated by the Mu C protein and initiates from four specific promoters.
- Understanding transcription termination is crucial for elucidating viral replication strategies.
Purpose of the Study:
- To characterize the transcription termination sites for the lys and P transcripts during phage Mu's late lytic cycle.
- To investigate the mechanisms underlying transcription termination, specifically Rho-dependence or independence.
Main Methods:
- Northern blot analysis to determine transcript lengths.
- S1 nuclease mapping to precisely locate transcript 3' ends.
- Plasmid-based assays to assess termination activity of cloned DNA fragments.
- Deletion analysis to identify essential sequences for termination.
Main Results:
- Lys and P transcripts were identified as approximately 7.6 kb and 6.3 kb, respectively.
- Termination sites were mapped to intergenic regions, dependent on the orientation of the invertible G segment.
- Cloned termination regions demonstrated efficient Rho-independent termination activity.
- Potential RNA stem-loop structures preceding the 3' ends were essential for termination.
Conclusions:
- The transcription termination sites for phage Mu lys and P transcripts function as Rho-independent terminators.
- Efficient termination relies on specific RNA secondary structures and, in some cases, upstream sequences.