The Structure of RecA Protein in Escherichia Coli.

我们开发了一种新的遗传方法，通过分析同源蛋白之间形成的嵌合体来寻找蛋白质的功能区。在携带大肠杆菌和铜绿假单胞菌recA基因的线性化质粒DNA中，通过分子内同源重组获得多组嵌合基因。将一株recBCsbcA菌株用于分离携带这些基因之间的重组子的质粒。对缺失recA基因并携带带有嵌合基因的质粒的大肠杆菌菌株的特性检测表明，在某些位置形成嵌合体会使RecA功能失活。通常情况下，所有与蛋白质某一区域连接的嵌合体都会失活一种功能。不是连接存在于特定位置的直接影响，而是来自不同物种的连接两侧区域的不匹配导致了失活。为了使嵌合蛋白质具有功能，蛋白质不同区域中的某些序列对必须来自同一亲本。发现了四对这样的序列：两对参与基因重组和抗紫外光照射的活性，另两对参与活性寡聚体的形成。由这些序列定义的区域位于蛋白质的环状区域。一对区域可以协作以形成功能性折叠结构。

We developed a novel genetic method for finding functional regions of a protein by the analysis of chimeras formed between homologous proteins. Sets of chimeric genes were made by intramolecular homologous recombination in a linearized plasmid DNA carrying both recA genes of Escherichia coli and Pseudomonas aeruginosa. A recBCsbcA strain of E. coli was used for isolation of plasmids carrying recombinants between these genes. Examination of properties of E. coli strains deleting the recA gene and carrying a plasmid with a chimeric gene shows that chimera formation at certain positions inactivates a RecA function. Frequently, all chimeras with a junction in a certain region of the protein inactivate a function. Rather than a direct effect of the presence of the junction at a particular position, mismatching of the regions both sides of the junction that are derived from the different species is responsible for the inactivation. For a chimeric protein to be functional, certain pairs of sequences in different regions of the protein must derive from the same parent. Four pairs of such sequences were found : two are involved in activities for genetic recombination and for resistance to ultraviolet light irradiation and the others in formation of active oligomera. Regions defined by these sequences are located in the looped regions of the protein. A pair of regions may cooperate to form a functional folded structure.