Truncated forms of IS911 transposase downregulate transposition.
IS911 naturally produces transposase (OrfAB) derivatives truncated at the C-terminal end (OrfAB-CTF) and devoid of the catalytic domain. A majority species, OrfAB*, was produced at higher levels at 42 degrees C than at 30 degrees C suggesting that it is at least partly responsible for the innate reduction in IS911 transposition activity at higher temperatures. An engineered equivalent of similar length, OrfAB[1-149], inhibited transposition activity in vivo or in vitro when produced along with full-length transposase. We isolated several point mutants showing higher activity than the wild-type IS911 at 42 degrees C. These fall into two regions of the transposase. One, located in the N-terminal segment of OrfAB, lies between or within two regions involved in protein multimerization. The other is located within the C-terminal catalytic domain. The N-terminal mutations resulted in reduced levels of OrfAB* while the C-terminal mutation alone appeared not to affect OrfAB* levels. Combination of N- and C-terminal mutations greatly reduced OrfAB* levels and transposition was concomitantly high even at 42 degrees C. The mechanism by which truncated transposase species are generated and how they intervene to reduce transposition activity is discussed. While transposition activity of these multiply mutated derivatives in vivo was resistant to temperature, the purified OrfAB derivatives retained an inherent temperature-sensitive phenotype in vitro. This clearly demonstrates that temperature sensitivity of IS911 transposition is a complex phenomenon with several mechanistic components. These results have important implications for the several other transposons and insertion sequences whose transposition has also been shown to be temperature-sensitive.
Gueguen
Erwan
E
Laboratoire de Microbiologie et de Génétique Moléculaire, UMR 5100 CNRS (Campus Paul Sabatier), 118 route de Narbonne, 31062 Toulouse Cedex 09, France.
Rousseau
Philippe
P
Duval-Valentin
Guy
G
Chandler
Mick
M
D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial; D004270:DNA, Circular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D012329:RNA, Bacterial; D012333:RNA, Messenger; D013696:Temperature; D019895:Transposases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004270:DNA, Circular; D029968:Escherichia coli Proteins; C085896:OrfB protein, E coli; D012329:RNA, Bacterial; D012333:RNA, Messenger; C473498:IS3 transposase; D019895:Transposases