Sub-terminal sequences modulating IS30 transposition in vivo and in vitro.
Inverted repeats of insertion sequences (ISs) are indispensable for transposition. We demonstrate that sub-terminal sequences adjacent to the inverted repeats of IS30 are also required for optimal transposition activity. We have developed a cell-free recombination system and showed that the transposase catalyses formation of a figure-of-eight transposition intermediate, where a 2 bp long single strand bridge holds the inverted repeat sequences (IRs) together. This is the first demonstration of the figure-of-eight structure in a non-IS3 family element, suggesting that this mechanism is likely more widely adopted among IS families. We show that the absence of sub-terminal IS30 sequences negatively influences figure-of-eight production both in vivo and in vitro. These regions enhance IR-IR junction formation and IR-targeting events in vivo. Enhancer elements have been identified within 51 bp internal to IRL and 17 bp internal to IRR. In the right end, a decanucleotide, 5'-GAGATAATTG-3', is responsible for wild-type activity, while in the left end, a complex assembly of repetitive elements is required. Functioning of the 10 bp element in the right end is position-dependent and the repetitive elements in the left end act cooperatively and may influence bendability of the end. In vitro kinetic experiments suggest that the sub-terminal enhancers may, at least partly, be transposase-dependent. Such enhancers may reflect a subtle regulatory mechanism for IS30 transposition.
Szabó
Mónika
M
Agricultural Biotechnology Center, 4 Szent-Györgyi Albert str., H-2100, Gödöllo, Hungary.
Kiss
János
J
Nagy
Zita
Z
Chandler
Michael
M
Olasz
Ferenc
F
D001483:Base Sequence; D001665:Binding Sites; D002474:Cell-Free System; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004270:DNA, Circular / Q000235:genetics; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D066298:In Vitro Techniques; D007700:Kinetics; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D012486:Salmonella typhimurium / Q000235:genetics; D020079:Terminal Repeat Sequences; D019895:Transposases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004270:DNA, Circular; D019895:Transposases