|Budget Amount *help
¥6,900,000 (Direct Cost : ¥6,900,000)
Fiscal Year 1991 : ¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1990 : ¥3,900,000 (Direct Cost : ¥3,900,000)
Transposon Tn3 encodes transposase, which promotes transposition of itself. We have previously purified Tn3 transposase and found that the transposase binds not only to the terminal inverted repeat (IR) sequences of Tn3 (38 base pairs in length) specifically, but also to any sequences nonspecifically. To analyze the domain structure of the Tn3 transposase protein, we cloned various DNA segments of the tnpA gene coding for transposase and fused with the beta-galactosidase gene (lacZ) in frame. We then purified the fusion proteins encoded by the truncated genes and examined their DNA-binding ability. We found that transposase had at least four domains, each of which exhibited the nonspecific DNA-binding ability. Interestingly, however, when the two of such domains located in the N-terminal region of transposase were combined, the region exhibited the IR-specific DNA-binding ability.
To analyze this IR-specific DNA-binding ability of Tn3 transposase further, we constructed mutants with mut
ations in various positions in the IR sequence and examined to see whether the IR mutants are bound by transposase.We found that the IR mutants with mutations within the region 13-18 (called A domain) are not bound by Tn3 transposase, while the IR mutants with mutations within the region 1-12 (called B domain) are bound by Tn3 transposase. This demonstrates that domain B is the region bound by transposase. We also examined the effect of mutations introduced in IR on transposition of Tn3 and found that the A and B domains were functionally distinct, but were required for transposition of Tn3. Domain A is supposed to be the region recognized by a cellular protein(s), which may be required for Tn3 transposition. A cellular protein of about 16 kDa in size, which could bind to IR,was detected by a gel-retardation assay. This protein may be a candidate protein that binds to domain A.
To understand the molecular mechanism of Tn3 transposition, we developed a system, in which transposition of Tn3 is reproduced in vitro using the bacterial cell extract only in the presence of transposase. The transposition reaction required Tn3 with the proper sequence and orientation of the IR sequences. The reaction also required DNA synthesis but not RNA synthesis by RNA polymerase. This is the first development of the in vitro system of transposition of Tn3 and is expected to be useful for the further analysis of the mechanism involved in transposition of Tn3.
(3)細胞破砕液に精製したトランスポゼ-スを加え、基質としてIRを末端に逆向きに持つものを使用することに依ってTn3の転移反応をin vitroで再現できた。この反応に於いて、ATP及びMg^<++>以外にDNA合成系が絶対的に要求されるがRNA合成系は必要でない。またDNA gyraseのインヒビターを加えると反応は進まない。 Less