Dynamics of a protein polymer: the assembly and disassembly pathways of the MuB transposition target complex.
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.
Greene
Eric C
EC
Laboratory of Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Mizuuchi
Kiyoshi
K
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D010583:Bacteriophage mu / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006868:Hydrolysis; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011108:Polymers; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D019895:Transposases / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; C069215:MuB protein, Enterobacteria phage Mu; D011108:Polymers; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004247:DNA; C114697:mu transposase; D019895:Transposases