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Phage Mu enlists the β-sliding clamp for late gene transcription
1Department of Molecular Biosciences and LaMontagne Center for Infectious Diseases, The University of Texas at Austin, Austin, TX 78712.
Abstract:
The phage Mu C protein (MuC), along with core RNA polymerase (RNAP) and σ70, is required for transcription of phage late genes. We found that overexpression of MuC was lethal in Escherichia coli and observed that host replication was overinitiating under these conditions. Suppressors of MuC lethality mapped to dnaA, diaA, and dnaX. DnaA initiates replication at oriC, assisted by DiaA. Reinitiation is prevented by hydrolysis of ATP-DnaA to ADP-DnaA by Hda and DnaN (β-sliding clamp or Clamp). DnaX, the tau/gamma subunit of Pol III, is part of the Clamp loader complex. Coexpression of hda and dnaN rescued MuC lethality. This result suggested that MuC was interfering with either Hda or DnaN. We noticed that MuC contains two near-consensus Clamp-binding motifs (CBM), one at the N terminus and one at the C-terminus. Changing the consensus residues of either CBM abolished MuC lethality, abrogated MuC-dependent transcription, and reduced plaque-forming units. Inactivation of a ts Clamp through temperature shift specifically inhibited MuC-dependent transcription but not σ70-dependent transcription. We show that MuC interacts with the Clamp to activate late gene transcription both in vivo and in vitro. This study demonstrates the involvement of the E. coli Clamp, a processivity factor essential for DNA replication, for transcription by E. coli RNAP. We observed that members of the Mor/MuC family of transcription factors all possess at least one CBM, suggesting that engaging the Clamp for transcription is likely to be more prevalent than hitherto recognized.
Insights
Phage Mu C protein (MuC) interacts with the E. coli beta-sliding clamp, essential for DNA replication, to activate phage transcription. This interaction is crucial for MuC
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Phage Mu C protein (MuC) is essential for transcription of phage late genes, requiring core RNA polymerase (RNAP) and σ70.
- Overexpression of MuC in Escherichia coli is lethal, causing host replication overinitiation.
- Suppression of MuC lethality involves genes regulating DNA replication initiation and termination (dnaA, diaA, dnaX).
Purpose of the Study:
- To investigate the molecular mechanism by which MuC causes lethality and activates transcription.
- To determine the interaction of MuC with host factors involved in DNA replication.
- To explore the broader implications of MuC-Clamp interaction for transcription regulation.
Main Methods:
- Genetic analysis of MuC lethality suppressors in E. coli.
- Site-directed mutagenesis of putative Clamp-binding motifs (CBMs) in MuC.
- In vivo and in vitro assays to assess MuC-dependent transcription and MuC-Clamp interaction.
- Temperature-sensitive mutant analysis of the beta-sliding clamp.
Main Results:
- MuC contains two functional CBMs essential for its lethality and transcriptional activity.
- Coexpression of Hda and DnaN (beta-sliding clamp) rescues MuC lethality, indicating MuC interferes with clamp function.
- MuC directly interacts with the E. coli beta-sliding clamp (DnaN) to activate phage late gene transcription.
- The beta-sliding clamp is essential for MuC-dependent transcription, but not for σ70-dependent transcription.
Conclusions:
- Phage MuC hijacks the essential DNA replication processivity factor, the beta-sliding clamp, to regulate transcription.
- This study reveals a novel mechanism where a replication factor is co-opted for transcriptional regulation.
- The presence of CBMs in other Mor/MuC family proteins suggests clamp engagement for transcription is a widespread phenomenon.
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