Congruence of in vivo and in vitro insertion patterns in hot E. coli gene targets of transposable element Mu: opposing roles of MuB in target capture and integration.
Phage Mu transposes promiscuously, employing protein MuB for target capture. MuB forms stable filaments on A/T-rich DNA, and a correlation between preferred MuB binding and Mu integration has been observed. We have investigated the relationship between MuB-binding and Mu insertion into hot and cold Mu targets within the Escherichia coli genome. Although higher binding of MuB to select hot versus cold genes was seen in vivo, the hot genes had an average A/T content and were less preferred targets in vitro, whereas cold genes had higher A/T values and were more efficient targets in vitro. These data suggest that A/T-rich regions are unavailable for MuB binding, and that A/T content is not a good predictor of Mu behavior in vivo. Insertion patterns within two hot genes in vivo could be superimposed on those obtained in vitro in reactions employing purified MuA transposase and MuB, ruling out the contribution of a special DNA structure or additional host factors to the hot behavior of these genes. While A/T-rich DNA is a preferred target in vitro, a fragment made up exclusively of A/T was an extremely poor target. A continuous MuB filament assembled along the A/T region likely protects it against the action of MuA. Our results suggest that MuB binds E. coli DNA in an interspersed manner utilizing local A/T richness, and facilitates capture of these bound regions by the transpososome. Actual integration events are then directed to sites that are in proximity to MuB filaments but are themselves free of MuB.
Ge
Jun
J
Section of Molecular Genetics and Microbiology and Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
Harshey
Rasika M
RM
D010583:Bacteriophage mu / Q000235:genetics / Q000378:metabolism; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000821:virology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D019895:Transposases / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; C069215:MuB protein, Enterobacteria phage Mu; D014764:Viral Proteins; D019895:Transposases