FUNCTION OF GROEL IN THE BACTERIAL CYTOPLASM

建议进行的研究旨在界定 均低聚双环14聚体复合体GroEL在折叠和组装中 细菌细胞质中新翻译的蛋白质。在.期间 在当前的项目期内，我们已经确定了一个明显的全球角色 线粒体基质中的同系物，Hsp60复合体，在介导 新进入细胞器的蛋白质的折叠和组装。我们 现在，我希望评估GroEL是否可以在 帮助新翻译的蛋白质达到它们的天然形式。这样的 观察表明有一种可能性：GroEL对于 细胞在任何温度下都能存活；该噬菌体组件 在几个非致命性GroEL突变体中受损；GroEL及其 协同组分GROE可介导几种细胞的体外复性 从变性剂中稀释的多肽。我们将评估GroEL的角色 在体内通过产生对温度敏感或对冷敏感的致命性 变种人。我们的方法将包括直接插入in的同源序列 将体外诱变的GroEL序列插入到大肠杆菌染色体中。 突变方法将包括定向密码子改变，掺杂 寡核苷酸诱变和丙氨酸扫描诱变。之后 转到不允许的温度，我们将比较野生型和 条件致死突变细胞：GroEL的分级行为 复合体；蛋白质翻译速率和翻译模式 产物；新翻译蛋白质的半衰期和溶解度； 几种蛋白质酶活性的翻译与获取 在不允许的温度下特别诱导，包括β- 半乳糖苷酶和半乳糖激酶。来自两个条件的GroEL复合体 致命性突变和生长缓慢的非致命性突变将被过度生产。 和纯化，以便在体外进行功能研究以区分 特定的缺陷，最终，一旦GroEL晶体结构 坚定的、结构-功能的理解。我们还将检查 其中多肽结合在GroEL上，无论是在其外表面还是 在环的空腔内，通过检查I扫描 十一碳偶联二氢叶酸的透射电子显微镜 与GroEL结合的还原酶。

The proposed studies are aimed at defining the function of the homooligomeric double ring 14mer complex, groEL, in folding and assembly of newly-translated proteins in the bacterial cytoplasm. During the current project period we have identified an apparently global role for a homologue in the mitochondrial matrix, hsp60 complex, in mediating folding and assembly of proteins newly-imported into the organelles. We now wish to assess whether groEL could have a similar general role for assisting newly-translated proteins to reach their native forms. such a possibility is suggested by observations: that groEL is essential for cell viability at all temperatures; that bacteriophage assembly is impaired in several nonlethal groEL mutants; and that groEL and its cooperating component groES can mediate refolding in vitro of several polypeptides diluted from denaturant. We will assess the role of groEL in vivo by producing temperature-sensitive or cold-sensitive lethal mutants. Our approach will involve direct homologous insertion of in vitro-mutagenized groEL sequences into the E. coli chromosome. Mutagenesis approaches will include directed codon changes, doped oligonucleotide mutagenesis, and alanine scanning mutagenesis. After shift to nonpermissive temperature, we will compare wild-type and conditional lethal mutant cells for: fractionation behavior of groEL complexes; rat of protein translation and pattern of translation products; half-life and solubility of the newly-translated proteins; translation and acquisition of enzyme activity of several proteins specifically induced at nonpermissive temperature, including beta- galactosidase and galactokinase. groEL complexes from both conditional lethal mutants and slow-growing nonlethal mutants will be overproduced and purified, to allow functional studies in vitro to distinguish specific defects, enabling ultimately, once a groEL crystal structure is determined, structure-function understandings. We will also examine where polypeptides are bound at groEL, whether at its outer surface or within the cavity of the rings, by examination i the scanning transmission electron microscope of undecagold-conjugated dihydrofolate reductase bound to groEL.