Characterization of two hypertransposing Tn5 mutants.
Transposition of Tn5 in Escherichia coli is regulated by two transposon-encoded proteins: transposase (Tnp), promoting transposition preferentially in cis, and the trans-acting inhibitor (Inh). Two separate transposase mutants were isolated that replace glutamate with lysine at position 110 (EK110) and at position 345 (EK345). The EK transposase proteins increase the Tn5 transposition frequency 6- to 16-fold in cis and enhance the ability of transposase to act in trans. The purified mutant transposase proteins interact with transposon outside end DNA differently from the wild-type protein, resulting in the formation of a novel complex in gel retardation assays. During characterization of the transposase proteins in the absence of inhibitor, we found that wild-type transposase itself has a transposition-inhibiting function and that this inhibition is reduced for the mutant proteins. We present a model for the regulation of Tn5 transposition, which proposes the existence of two transposase species, one cis-activating and the other trans-inhibiting. The phenotype of the EK transposase mutants can be explained by a shift in the ratio of these two species.
Wiegand
T W
TW
Department of Biochemistry, University of Wisconsin-Madison 53706.
Reznikoff
W S
WS
D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics* / Q000502:physiology; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005809:Genes, Regulator / Q000235:genetics*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D019895:Transposases
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D009713:Nucleotidyltransferases; D019895:Transposases