The Structure of RecA Protein in Escherichia Coli.

我们开发了一种新的遗传方法，通过分析同源蛋白质之间形成的嵌合体来寻找蛋白质的功能区域。通过在携带大肠杆菌和铜绿假单胞菌的recA基因的线性化质粒DNA中进行分子内同源重组来制备嵌合基因组。recBCsbcA菌株是E.大肠杆菌用于分离携带这些基因之间的重组体的质粒。对E.缺失recA基因并携带具有嵌合基因的质粒的大肠杆菌菌株显示，在某些位置形成嵌合体使RecA功能失活。通常情况下，所有在蛋白质的特定区域中具有连接的嵌合体都发挥作用。而不是在特定位置处的接合点的存在的直接影响，源自不同物种的接合点两侧的区域的错配是导致失活的原因。为了使嵌合蛋白具有功能，蛋白质不同区域中的某些序列对必须来自相同的亲本。发现了四对这样的序列：两个参与遗传重组和抗紫外线照射的活动，其他的参与活性寡聚体的形成。由这些序列限定的区域位于蛋白质的环状区域中。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.