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Specific nicking at the 3′ ends of the terminal inverted repeat sequences in transposon Tn 3 by transposase and an E. coli protein ACP
Author(s) -
Maekawa Takafumi,
Yanagihara Katsuhiko,
Ohtsubo Eiichi
Publication year - 1996
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1046/j.1365-2443.1996.d01-221.x
Subject(s) - transposase , biology , transposable element , inverted repeat , escherichia coli , transposition (logic) , microbiology and biotechnology , plasmid , insertion sequence , biochemistry , dna , gene , mutant , linguistics , philosophy , genome
Background : Tn 3 , a bacterial transposon, carries tnpA gene encoding transposase which is essential for its transposition. The transposition of Tn 3 has been reproduced in vitro in a cell extract containing transposase by using a plasmid carrying mini‐Tn 3 as the donor and another plasmid as the target. Transposase has the ability to bind to the 38‐bp terminal inverted repeats (IRs) of Tn 3 . The molecular mechanism of the initiation step of the Tn3 transposition reaction promoted by the transposase has, however, not been understood. Results : We found that nicking occurred efficiently in the cell‐free system at each of the 3′ ends of the IRs of mini‐Tn 3 in the closed circular or linear donor molecules. The nicking reaction required transposase and Mg 2+ , but did not require ATP, an ATP‐regenerating system, dNTPs and polyvinyl alcohol, which were the requirements for the transposition reaction. By using the nicking assay employed here, transposase was purified almost to homogeneity. Gel filtration and sedimentation analyses indicate that transposase forms a dimer in a solution containing 0.5 m NaCl. The nicking activity of the purified transposase was weak and was found to be stimulated by a host factor. The nicking stimulation factor was subsequently purified and found to be ACP, an Escherichia coli acyl carrier protein. Conclusions : Nicking occurred efficiently at the 3′ ends of mini‐Tn 3 in the reaction mixture containing transposase and ACP. ACP is known to act as a factor which modulates enzymes that are involved in several biological processes either in the acylated or unacylated form. ACP may also modulate transposase to initiate the transposition reaction with nicking at the 3′ ends of Tn 3 .