Vogel J L, Li Z J, Howe M M, Toussaint A, Higgins N P
Department of Biochemistry, University of Alabama, Birmingham 35294.
J Bacteriol. 1991 Oct;173(20):6568-77. doi: 10.1128/jb.173.20.6568-6577.1991.
Phage Mu's c gene product is a cooperative regulatory protein that binds to a large, complex, tripartite 184-bp operator. To probe the mechanism of repressor action, we isolated and characterized 13 phage mutants that cause Mu to undergo lytic development when cells are shifted from 30 to 42 degrees C. This collection contained only four mutations in the repressor gene, and all were clustered near the N terminus. The cts62 substitution of R47----Q caused weakened specific DNA recognition and altered cooperativity in vitro. A functional repressor with only 63 amino acids of Mu repressor fused to a C-terminal fragment of beta-galactosidase was constructed. This chimeric protein was an efficient repressor, as it bound specifically to Mu operator DNA in vitro and its expression conferred Mu immunity in vivo. A DNA looping model is proposed to explain regulation of the tripartite operator site and the highly cooperative nature of repressor binding.
噬菌体Mu的c基因产物是一种协同调节蛋白，它能结合到一个大的、复杂的、由三个部分组成的184碱基对的操纵子上。为了探究阻遏物的作用机制，我们分离并鉴定了13个噬菌体突变体，当细胞从30℃转移到42℃时，这些突变体会导致Mu进行裂解发育。这个集合中阻遏物基因只有四个突变，并且都聚集在N端附近。R47→Q的cts62替换导致体外特异性DNA识别减弱和协同性改变。构建了一种功能性阻遏物，它仅由63个氨基酸的Mu阻遏物与β-半乳糖苷酶的C端片段融合而成。这种嵌合蛋白是一种有效的阻遏物，因为它在体外能特异性结合Mu操纵子DNA，并且其表达在体内赋予了Mu免疫性。我们提出了一个DNA环化模型来解释三联体操纵子位点的调控以及阻遏物结合的高度协同性。
