STUDY OF FUNCTIONS INVOLVED IN GENETIC RECOMBINATION

DNA在大肠杆菌中的超螺旋水平是由 DNA旋转酶的DNA超卷曲活性和DNA松弛活性 拓扑异构酶I.我们已经证明了这两种酶的合成都是在 由超级卷曲程度控制的旋转；例如，如果单元格的 DNA不够超螺旋，DNA旋转酶较多，拓扑异构酶I较少 这样，超级卷曲就会增加到一个更正常的水平。我们 最近研究了这两种旋转酶的基因表达 亚基(gyrA和gyrB)，以了解这种控制是如何工作的。删除 对两个启动子的分析表明，DNA所需的区域 松弛诱导表达很少，不超过21个碱基对。 这两个启动子也是非典型的，因为下游序列 更重要的是，上游序列比正常情况下更重要 大肠杆菌启动子。 我们已经测定了大肠杆菌gyrB基因的DNA序列，并使用了 结果定位于天然蛋白质中产生一种 细胞提取液中部分活性的Gyr B片段。该片段是 分子的C末端的一半。我们还将此序列与 枯草杆菌gyrB基因的同源性。大肠杆菌Gyr B蛋白(MW) 90,000)可以被证明含有一个额外的近200个氨基酸的环。 DNA旋转酶依赖于DNA的ATPase活性的研究表明，DNA A2B2酶的两个半分子中的结合部位必须填充 才能发生三磷酸腺苷的水解。短链DNA和三磷酸腺苷的结合 两者都被证明是高度合作的。我们做了一件粗略的事 基于电二向色性的酶-DNA复合体空间模型 实验。

The level of DNA superhelicity in E. coli is set by the balance of the DNA-supercoiling activity of DNA gyrase and the DNA-relaxing activity of topoisomerase I. We have shown that the synthesis of both enzymes is in turn controlled by the level of supercoiling; if, for example, the cell's DNA is not supercoiled enough, more DNA gyrase and less topoisomerase I are made, so that supercoiling then increases toward a more normal level. We have recently studied the expression of the genes for the two gyrase subunits (gyrA and gyrB) so as to learn how this control works. A deletion analysis of both promoters shows that the region needed for DNA relaxation-induced expression is very small, no more than 21 base pairs. Both of these promoters are also atypical in that down-stream sequences are more important, and upstream sequences less important, than is normal for E. coli promoters. We have determined the DNA sequence of the E. coli gyrB gene and used the results to locate within the native protein the cleavage that produces a partly active Gyr B fragment in cell extracts. The fragment is the C-terminal half of the molecule. We have also compared this sequence with that of the gyrB gene of B. subtilis. The E. coli Gyr B protein (MW 90,000) can be shown to contain one extra loop of almost 200 amino acids. A study of the DNA-dependent ATPase activity of DNA gyrase shows that DNA binding sites in both half-molecules of the A2B2 enzyme must be filled before ATP hydrolysis can occur. Binding of short DNA chains and of ATP have both been shown to be highly cooperative. We have made a rough spatial model of the enzyme-DNA complex based on electro-dichroism experiments.