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Temperature-sensitive mutations in the bacteriophage Mu c repressor locate a 63-amino-acid DNA-binding domain
Abstract:
Phage Mu's c gene product is a cooperative regulatory protein that binds to a large, complex, tripartite 184-bp operator. To probe the mechanism of repressor action, we isolated and characterized 13 phage mutants that cause Mu to undergo lytic development when cells are shifted from 30 to 42 degrees C. This collection contained only four mutations in the repressor gene, and all were clustered near the N terminus. The cts62 substitution of R47----Q caused weakened specific DNA recognition and altered cooperativity in vitro. A functional repressor with only 63 amino acids of Mu repressor fused to a C-terminal fragment of beta-galactosidase was constructed. This chimeric protein was an efficient repressor, as it bound specifically to Mu operator DNA in vitro and its expression conferred Mu immunity in vivo. A DNA looping model is proposed to explain regulation of the tripartite operator site and the highly cooperative nature of repressor binding.
Insights
Researchers studied bacteriophage Mu
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Bacteriophage Mu's c gene product is a regulatory protein controlling viral replication.
- This protein binds to a complex, tripartite operator DNA sequence.
- Understanding repressor-operator interactions is key to phage regulation.
Purpose of the Study:
- To investigate the mechanism of bacteriophage Mu repressor action.
- To identify mutations affecting repressor function and DNA binding.
- To elucidate the DNA binding and regulatory properties of the Mu c repressor.
Main Methods:
- Isolation and characterization of temperature-sensitive Mu phage mutants.
- Site-directed mutagenesis and protein expression.
- In vitro DNA binding assays and in vivo Mu immunity tests.
- Construction of a chimeric repressor protein.
Main Results:
- Four mutations in the repressor gene were identified, clustered near the N-terminus.
- The cts62 mutation (R47Q) reduced DNA binding affinity and altered cooperativity.
- A functional chimeric repressor (63 aa Mu repressor fused to beta-galactosidase) was created.
- This chimera bound specifically to Mu operator DNA and conferred immunity.
Conclusions:
- The N-terminus of the Mu repressor is critical for its function.
- A DNA looping model can explain the tripartite operator regulation and cooperative binding.
- Chimeric proteins can retain repressor activity, aiding functional studies.