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Characterization of the cts4 repressor mutation in transposable bacteriophage Mu
Philippe Rousseau1, Jamal E Laachouch, Mick Chandler
1Laboratoire de Microbiologie et de Génétique Moléculaire, CNRS-université Toulouse III, France.
Abstract:
Mucts4 was isolated more than 30 years ago and was the first available thermoinducible derivative of transposable phage Mu. We have characterized the cts4 mutation and the corresponding mutant protein. Contrary to previously characterized thermoinducible Mu prophages (e.g., Mucts62), Mucts4 lysogenizes at reduced frequency even at 30 degrees C. The cts4 mutation (Leu129Val) was located in this central repressor region. The cts4 protein was thermosensitive for operator DNA binding in vitro. Temperature-dependent changes in protein-protein cross-linking patterns in the absence of DNA were detected for purified wild type, cts62 and cts4 repressor proteins. The cts4 protein exhibited a subtly different electrophoretic profile, which became more marked at higher temperatures, from both the wild type and cts62. In addition the cts4 repressor generated a significantly different pattern of binding to DNA fragments carrying the early operator region. Consistent with the predicted involvement of the central leucine-rich region of the Mu repressor in the formation of multimeric forms, the cts4 mutation thus appeared to affect protein-protein interactions.
Insights
The Mucts4 phage repressor protein shows thermosensitive DNA binding and altered protein interactions, impacting its ability to lysogenize. This study characterizes the cts4 mutation
Area of Science:
- Molecular Biology
- Bacteriology
- Genetics
Background:
- Mucts4 is a thermoinducible derivative of transposable phage Mu, isolated over 30 years ago.
- It was the first available thermoinducible derivative of transposable phage Mu.
- Previously characterized thermoinducible Mu prophages, such as Mucts62, exhibit different lysogenization characteristics.
Purpose of the Study:
- To characterize the cts4 mutation and its corresponding mutant protein.
- To understand the molecular basis of the thermosensitivity and altered function of the Mucts4 repressor.
- To investigate the effect of the cts4 mutation on repressor-DNA and repressor-repressor interactions.
Main Methods:
- Characterization of the cts4 mutation (Leu129Val) within the repressor region.
- In vitro assessment of thermosensitive operator DNA binding of the cts4 repressor.
- Analysis of temperature-dependent protein-protein cross-linking patterns.
- Electrophoretic analysis of wild-type, Mucts62, and Mucts4 repressor proteins.
- Investigation of Mucts4 repressor binding to DNA fragments containing the early operator region.
Main Results:
- The Mucts4 repressor exhibits thermosensitive operator DNA binding in vitro.
- Temperature-dependent alterations in protein-protein cross-linking were observed for Mucts4 repressor.
- The Mucts4 repressor displayed a distinct electrophoretic profile compared to wild-type and Mucts62 repressors, especially at higher temperatures.
- Mucts4 repressor showed altered binding patterns to DNA fragments with the early operator region.
- The cts4 mutation appears to affect protein-protein interactions, potentially within the leucine-rich region.
Conclusions:
- The cts4 mutation (Leu129Val) in the Mu phage repressor leads to thermosensitive DNA binding and altered protein-protein interactions.
- These molecular defects contribute to the reduced lysogenization frequency of Mucts4, even at permissive temperatures.
- The findings provide insights into the structure-function relationships of the Mu repressor and its role in phage-Mu regulation.