Wiegand T W, Reznikoff W S
Department of Biochemistry, University of Wisconsin-Madison 53706.
J Mol Biol. 1994 Jan 14;235(2):486-95. doi: 10.1006/jmbi.1994.1008.
Transposition of Tn5 requires the binding of the transposase protein to the transposon outside end (OE) DNA sequences. Transposase mutants that increase the transposition frequency result in the formation of two distinct transposase/OE DNA complexes, observed by gel retardation analysis. The slower migrating complex I, also formed by wild-type transposase, contains protein oligomers of transposase and transposase related proteins. The faster migrating, novel complex II is caused by the binding of monomeric, proteolytic transposase fragments gamma and delta that have lost the carboxy-terminus of the protein. Transposase gamma and delta bind OE DNA with a high apparent affinity but are unable to promote transposition in vivo. We propose that the transposase protein is functionally unstable and can undergo a conformational change that reduces the activity but protects the protein from proteolysis. The transposase mutants favor the more active but proteolytically hypersensitive protein conformation.
Tn5 的转座需要转座酶蛋白与转座子外侧末端（OE）DNA 序列结合。通过凝胶阻滞分析观察到，增加转座频率的转座酶突变体会导致形成两种不同的转座酶/OE DNA 复合物。迁移较慢的复合物 I 也由野生型转座酶形成，它包含转座酶的蛋白质寡聚体和转座酶相关蛋白。迁移较快的新型复合物 II 是由已失去蛋白质羧基末端的单体、蛋白水解转座酶片段γ和δ结合所导致的。转座酶γ和δ以高表观亲和力结合 OE DNA，但在体内无法促进转座。我们提出转座酶蛋白在功能上不稳定，会发生构象变化，这种变化会降低活性但能保护蛋白质不被蛋白水解。转座酶突变体有利于形成活性更高但对蛋白水解更敏感的蛋白质构象。
