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Dynamics of a protein polymer: the assembly and disassembly pathways of the MuB transposition target complex
Eric C Greene1, Kiyoshi Mizuuchi
1Laboratory of Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Abstract:
MuB assembles into a polymer on DNA in the presence of ATP and is directly involved in the selection of an appropriate site on the Escherichia coli chromosome for the insertion of the bacteriophage Mu genome. We have developed an assay using fluorescently tagged proteins to monitor the polymeric state of MuB via fluorescence resonance energy transfer. We show that polymer assembly is initiated by the formation of an ATP-MuB complex. MuB then self-associates into a protomer before binding to DNA. Upon binding to DNA, a dramatic increase in energy transfer is observed, suggesting a conformational change within MuB. Polymer disassembly is much slower than assembly and is greatly stimulated by the MuA transposase. Additionally, MuB is readily exchanged between polymers, and ATP hydrolysis is directly coupled to polymer disassembly. Our data support a model in which a combination of rapid polymer assembly, MuA-mediated disassembly, followed by rapid reassembly of the polymer allows MuB to sample multiple DNA targets until an appropriate site is located for the insertion of the bacteriophage genome.
Insights
Bacteriophage Mu protein B (MuB) forms polymers on DNA to help select insertion sites. ATP fuels assembly, while MuA protein speeds up disassembly, allowing MuB to efficiently find targets.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Bacteriophage Mu utilizes the MuB protein for site-specific DNA integration.
- Understanding MuB's assembly and disassembly is crucial for elucidating bacteriophage Mu's life cycle.
Purpose of the Study:
- To investigate the polymerization dynamics of MuB protein on DNA.
- To elucidate the role of ATP and MuA protein in MuB polymer assembly and disassembly.
Main Methods:
- Fluorescence resonance energy transfer (FRET) assay with fluorescently tagged MuB.
- Monitoring MuB polymerization state in response to ATP and DNA binding.
Main Results:
- MuB polymer assembly is initiated by ATP-MuB complex formation and is rapid.
- DNA binding induces a conformational change in MuB, increasing FRET.
- Polymer disassembly is slow but stimulated by MuA transposase; ATP hydrolysis drives disassembly.
- MuB proteins are readily exchanged between polymers.
Conclusions:
- MuB polymer dynamics, including rapid assembly and MuA-stimulated disassembly, facilitate target DNA sampling.
- ATP hydrolysis is coupled to MuB polymer disassembly, regulating the selection process.
- This mechanism ensures efficient and accurate insertion of the bacteriophage Mu genome.