Evidence that the cis preference of the Tn5 transposase is caused by nonproductive multimerization.
The transposase (Tnp) of the bacterial transposon Tn5 acts 50- to 100-fold more efficiently on elements located cis to the site of its synthesis compared with those located in trans. In an effort to understand the basis for this cis preference, we have screened for Tnp mutants that exhibit increased transposition activity in a trans assay. Two mutations in the carboxyl terminus were isolated repeatedly. The EK345 mutation characterized previously increases Tnp activity eightfold both in cis and in trans. The novel LP372 mutation, however, increases Tnp activity 10-fold specifically in trans. Combining both mutations increases Tnp activity 80-fold. Interestingly, the LP372 mutation maps to a region shown previously to be critical for interaction with Inh, an inhibitor of Tn5 transposition, and results in reduced inhibition activity by both Tnp and Inh. Tnp also inhibits Tn5 transposition in trans, and this has been suggested to occur by the formation of inactive Tnp multimers. Because Inh and (presumably) Tnp inhibit Tn5 transposition by forming defective multimers with Tnp, the inhibition defect of the trans-active LP372 mutant suggests that the cis preference of Tnp may also be attributable to nonproductive Tnp-Tnp multimerization. In addition, we show that increasing the synthesis of EK345/LP372 Tnp, but not wild-type Tnp, leads to very high levels of transposition, presumably because this altered Tnp is defective in the inhibitory activity of the wild type protein.
Weinreich
M D
MD
Department of Biochemistry, University of Wisconsin, Madison 53706.
Gasch
A
A
Reznikoff
W S
WS
D001483:Base Sequence; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D015533:Transcriptional Activation; D019895:Transposases
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D009713:Nucleotidyltransferases; D019895:Transposases