STUDY OF A RIBONUCLEASE AND ITS INHIBITOR FROM BACILLUS AMYLOLIQUEFACIENS

两种蛋白质，芽孢杆菌RNA酶，芽孢杆菌的胞外核糖核酸酶 解淀粉菌及其细胞内抑制剂芽孢杆菌RNA酶抑制剂 作为研究蛋白质折叠和蛋白质-蛋白质的模型系统 交互.芽孢杆菌RNA酶是核糖核酸酶的同源群之一 在原核生物和真核生物中都有发生。重组DNA技术 有三个主要目的：（1）促进生产 野生型和突变型蛋白质;（2）检查结构和 基因的控制序列;和（3）在基因中进行特异性改变。 序列来测试折叠理论和探测barnase-barstar 互动这两种蛋白质现在都可以从重组基因中获得 在大肠在大肠杆菌中，芽孢杆菌RNA酶抑制剂的表达抵消了 芽孢杆菌RNA酶表达这两种蛋白质及其复合物的结构 是已知的。1.5 ℃结构的新晶体学解 埃分辨率提供了新的见解。例如几 可以看到在barnase-barstar界面上的水分子 已经被束缚在自由的barnase上了几对巴纳斯-巴斯塔尔夫妇 在界面中的互补突变，通过体内 选择性技术，已结晶和结构immiment。 Barstar还抑制来自链霉菌菌株的一组RNA酶。这些 酶与arnase的序列同一性仅为 百分之二十五其中，认同度从40%到70%不等。的结构 其中两种，RNA酶Sa和St，是从非重组的工作中已知的。 材料我们现在有了第三种核糖核酸酶Sa 2的结构， 和巴斯塔尔有关系核糖核酸酶基因的重叠片段 St，这是很难抑制barstar，已被克隆，使我们能够 以纠正其公布的氨基酸序列中的几个错误。基因 已克隆并测序了芽孢杆菌RNA酶抑制剂的Sa 2同源物。这 蛋白质抑制所有链霉菌酶，但显然不是 barnase 芽孢杆菌RNA酶基因的可控致死性已被用于 为了有利于产生选择性通用质粒克隆 向量

Two proteins, barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens, and barstar, its intracellular inhibitor, are used as a model system for the study of protein folding and protein-protein interactions. Barnase is one of an homologous group of ribonucleases ocurring in both prokaryotes and eukaryotes. Recombinant DNA techniques are being applied with three major aims: (1) to facilitate production of wild type and mutant proteins; (2) to examine the structural and contro sequences of the genes; and (3) to make specific changes in the sequences to test theories of folding and to probe the barnase-barstar interaction. Both proteins can now be obtained from recombinant genes in E. coli where expression of barstar counters the lethal effect of barnase expression. The structures of both proteins and their complex are known. A new crystallographic solution of the structure at 1.5 angstroms resolution has provided new insights. For example, several water molecules seen in the barnase-barstar interface can be seen already bound to free barnase. Several barnase-barstar pairs having complementary mutations in the interface, obtained by an in vivo selective technique, have been crystallized and structures are immiment. Barstar also inhibits a group of RNases from Streptomyces strains. These enzymes are distantly related to arnase with a sequence identity of only 25%. Among themselves, identify ranges from 40% to 70%. The structures of two, RNases Sa and St, are known from work on nonrecombinant material. We now have the structure of a third, RNase Sa2, both alone and in complex with barstar. Overlapping fragments of the gene for RNase St, which is poorly inhibited by barstar, have been cloned, allowing us to correct several errors in its published amino acid sequence. The gene for Sa2 homolog of barstar has now been cloned and sequenced. This protein inhibits all the Streptomyces enzymes but apparently not barnase. The controllable lethality of the barnase gene has been used to advantage in producing a selective general purpose plasmid cloning vector