Wu Z, Chaconas G
Department of Biochemistry, University of Western Ontario, London, Canada.
J Biol Chem. 1994 Nov 18;269(46):28829-33.
Mu A protein, the 75-kDa phage transposase, consists of three domains: a 30-kDa NH2 terminus, a 35-kDa central domain, and a 10-kDa COOH terminus (Nakayama, C., Teplow, D. B., and Harshey, R. M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1809-1813). Genetic and biochemical experiments have demonstrated that the COOH-terminal domain must be present for functional interaction with Mu B protein. To further investigate the COOH-terminal domain of Mu A, we fused this 89-amino acid region to the glutathione S-transferase gene to facilitate subsequent expression and purification. We show that either the glutathione S-transferase-peptide fusion protein or the COOH-terminal peptide severed from glutathione S-transferase is active in Mu B interaction. Addition of the COOH-terminal domain to the in vitro strand transfer reaction inhibits intermolecular strand transfer by a mechanism previously characterized for intact Mu A protein (Baker, T. A., Mizuuchi, M., and Mizuuchi, K. (1991) Cell 65, 1003-1013), although the COOH-terminal domain is 70 times less effective than intact Mu A. The transient interaction between the COOH-terminal domain and Mu B does not inhibit Mu B stimulation of the strand cleavage and intramolecular strand transfer activity of Mu A. Deletion analysis has shown that the last 36 amino acids are sufficient for interaction with Mu B, but that removal of as few as 4 amino acids from the COOH terminus renders the peptide inactive. The recovery of an active COOH-terminal domain of Mu A will facilitate future structure/function studies of the Mu transposase.
Mu A蛋白，即75 kDa的噬菌体转座酶，由三个结构域组成：一个30 kDa的NH2末端、一个35 kDa的中央结构域和一个10 kDa的COOH末端（中山，C.，特普洛，D. B.，和哈谢，R. M.（1987年）《美国国家科学院院刊》84，1809 - 1813）。遗传学和生物化学实验表明，COOH末端结构域对于与Mu B蛋白的功能相互作用是必需的。为了进一步研究Mu A的COOH末端结构域，我们将这个89个氨基酸的区域与谷胱甘肽S - 转移酶基因融合，以促进后续的表达和纯化。我们发现，谷胱甘肽S - 转移酶 - 肽融合蛋白或从谷胱甘肽S - 转移酶上切割下来的COOH末端肽在与Mu B相互作用中均具有活性。将COOH末端结构域添加到体外链转移反应中，会通过一种先前已针对完整Mu A蛋白进行过表征的机制抑制分子间链转移（贝克，T. A.，水内，M.，和水内，K.（1991年）《细胞》65，1003 - 1013），尽管COOH末端结构域的效果比完整的Mu A低70倍。COOH末端结构域与Mu B之间的瞬时相互作用并不抑制Mu B对Mu A的链切割和分子内链转移活性的刺激作用。缺失分析表明，最后36个氨基酸足以与Mu B相互作用，但从COOH末端移除少至4个氨基酸就会使该肽失去活性。Mu A活性COOH末端结构域的获得将有助于未来对Mu转座酶的结构/功能研究。
