STUDY OF A RIBONUCLEASE AND ITS INHIBITOR FROM BACILLUS AMYLOLIQUEFACIENS

芽孢杆菌的胞外核糖核酸酶Barnase 淀粉分解酶和其胞内抑制物Barstar被使用 作为蛋白质折叠和蛋白质-蛋白质研究的模型系统 互动。Barnase是核糖核酸酶的同源基团之一 在原核生物和真核生物中都存在。重组DNA技术 应用有三个主要目的：(1)促进生产 野生型和突变型蛋白；(2)检测结构和 基因的控制序列；以及(3)对基因的 检验折叠理论和探测Barnase-Barstar的序列 互动。这两种蛋白质现在都可以从重组基因中获得。 在大肠杆菌中，Barstar的表达抵消了BAR的致死作用 Barnase表达。蛋白质及其复合体的结构 都是已知的。一种新的1.5℃结构的晶体溶液 Angstroms解析法提供了新的见解。例如，有几个 在棒石-棒星界面上可以看到水分子。 已经被绑起来释放藤壶了。几个藤壶-杠星对具有 界面上的互补突变，通过体内的 选择性技术，已经结晶，结构即将到来。 Barstar还抑制链霉菌菌株的一组核糖核酸酶。这些 酶与Arnase有较远的亲缘关系，其序列同一性仅为 25%。其中，认同度从40%到70%不等。这些结构 其中两个，RNase Sa和St，是从非重组的工作中得知的 材料。我们现在有了第三个结构，RNaseSA2，两者都是单独的 在与巴斯塔尔的综合体中。核糖核酸酶基因的重叠片段 被Barstar抑制得很差的ST已经被克隆，使我们能够 纠正其公布的氨基酸序列中的几个错误。基因 对于SA2，Barstar的同源基因现已被克隆和测序。这 蛋白质能抑制链霉菌的所有酶，但显然不能 巴纳萨斯。Barnase基因的可控致死性已被利用 有利于生产具有选择性的通用质粒克隆 矢量

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