GENETIC ANALYSIS OF HEAT SHOCK PROTEIN FUNCTION

这项研究的长期目标是更好地 了解大肠杆菌热的分子功能 休克蛋白、DNAK和GroELS。这些蛋白质属于一种 进化上高度保守的一类分子，称为 催化多肽链折叠的伴侣蛋白 变成它们的最终构象。蛋白质折叠催化有 已经被证明在各种基本的细胞中都很重要 膜转位、低聚物组装等过程 蛋白质复合体与细胞成分的抗病毒作用 应力引起的损伤。 因为这些热休克基因在单个 复制，大肠杆菌提供了一个很好的系统来研究这些 蛋白质。将采取一种传统的遗传方法，并将 包括在dNAK和groELS中产生突变，这些突变 影响它们的伴侣活性。筛查程序将是 利用新发现的DNAK的能力和 GroEL将促进Lamb-LacZ杂交蛋白从 细胞质。高效输出这些LacZ杂交种的细胞 成为表型Lac-。Lac+返回体，将被分离 并应代表不再拥有伴侣的突变体 活动。新的dnaK和groELS突变体的特征是 与已知等位基因和DNA序列的表型比较 分析。DNAK和GroEL的关联能力 体内的Lamb-LacZ杂交蛋白也将被研究。在……里面 此外，DNAK和GroELS在其他大肠杆菌出口中的作用 蛋白质将通过耗尽细胞中的这些 陪伴者。新的突变体应该被证明在确定 这些热休克蛋白在细胞过程中的作用，如 蛋白质出口。此外，羔羊-Lacz的Lac表型 融合菌株将被用于寻找其他蛋白质，来自一种 多种生物体，它们具有伴侣活性。 对细胞的分子功能的理解 伴侣蛋白应该提供对各种细胞的洞察 进程。它们可能会被证明对工程师E很有用。Coli到组装 具有重要医学和农业价值的蛋白质复合体 蛋白质复合体。此外，蛋白质显然也是 感染性免疫反应中的主要抗原决定因素和 在免疫监测中可能很重要。新技术的隔绝 来自大肠杆菌的伴侣蛋白突变体应该被证明在 理解这种重要的分子功能的多样性 分子的类别。

The long-term objective of this research is to better understand the molecular functions of the Escherichia coli heat shock proteins, DnaK and GroELS. These proteins belong to a highly evolutionarily conserved class of molecules called chaperonins, which catalyze the folding of polypeptide chains into their final conformations. Protein folding catalysis has been shown to be important in a variety of fundamental cellular processes, such as membrane translocation, assembly of oligomeric protein complexes and protection of cellular components against stress-induced damage. Because of the presence of these heat shock genes in single copy, E. coli provides an excellent system to study these proteins. A traditional genetic approach will be taken and will include the generation of mutations in both dnaK and groELS which affect their chaperonin active. A screening procedure will be employed which exploits the newly discovered ability of DnaK and GroEL to facilitate the export of LamB-LacZ hybrid proteins from the cytoplasm. Cells which efficiently export these LacZ hybrids become phenotypically Lac-. Lac+ revertants, will be isolated and should represent mutants which no longer possess chaperonin activity. New dnaK and groELS mutants will be characterized by phenotypic comparison with known alleles and by DNA sequence analysis. The ability of DnaK and GroEL to associate with LamB-LacZ hybrid proteins in vivo will also be investigated. In addition, the role of DnaK and GroELS in export of other E. coli proteins will be addressed by depleting the cells of these chaperonins. New mutants should prove useful in determining the roles of these heat shock proteins in cellular processes, such as protein export. In addition, the Lac phenotype of lamB-lacZ fusion strains will be used to search for other proteins, from a variety of organisms, which have chaperonin activity. An understanding of the molecular functioning of the chaperonins should provide insights into a variety of cellular process. They may prove useful to engineer E . coli to assembly protein complexes of medically and agriculturally important protein complexes. In addition, proteins are also apparently major antigenic determinants in infectious immune responses and may be important in immune surveillance. The isolation of new chaperonin mutants from E. coli should prove valuable in understanding the diverse molecular functions of this important class of molecules.