Interactions of phage Mu enhancer and termini that specify the assembly of a topologically unique interwrapped transpososome.
The higher-order DNA-protein complex that carries out the chemical steps of phage Mu transposition is organized by bridging interactions among three DNA sites, the left (L) and right (R) ends of Mu, and an enhancer element (E), mediated by the transposase protein MuA. A subset of the six subunits of MuA associated with their cognate sub-sites at L and R communicate with the enhancer to trigger the stepwise assembly of the functional transpososome. The DNA follows a well-defined path within the transpososome, trapping five supercoil nodes comprising two E-R crossings, one E-L crossing and two L-R crossings. The enhancer is a critical DNA element in specifying the unique interwrapped topology of the three-site LER synapse. In this study, we used multiple strategies to characterize Mu end-enhancer interactions to extend, modify and refine those inferred from earlier analyses. Directed placement of transposase subunits at their cognate sub-sites at L and R, analysis of the protein composition of transpososomes thus obtained, and their characterization using topological methods define the following interactions. R1-E interaction is essential to promote transpososome assembly, R3-E interaction contributes to the native topology of the transpososome, and L1-E and R2-E interactions are not required for assembly. The data on L2-E and L3-E interactions are not unequivocal. If they do occur, either one is sufficient to support the assembly process. Our results are consistent with two R-E and perhaps one L-E, being responsible for the three DNA crossings between the enhancer and the left and right ends of Mu. A 3D representation of the interwrapped complex (IW) obtained by modeling is consistent with these results. The model reveals straightforward geometric and topological relationships between the IW complex and a more relaxed enhancer-independent V-form of the transpososome assembled under altered reaction conditions.
Yin
Zhiqi
Z
Section of Molecular Genetics and Microbiology and Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
Suzuki
Asaka
A
Lou
Zheng
Z
Jayaram
Makkuni
M
Harshey
Rasika M
RM
D010583:Bacteriophage mu / Q000201:enzymology* / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D019895:Transposases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016662:Virus Integration
D021122:Protein Subunits; D019895:Transposases