MECHANISM OF PROTEIN LOCALIZATION IN ESCHERICHIA COLI

大肠杆菌中蛋白质定位的机制

• 批准号: 2181197
• 负责人: DONALD B. OLIVER
• 金额: $ 21.62万
• 依托单位: WESLEYAN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2181197
• 关键词: Escherichia coli; adenosinetriphosphatase; bacterial proteins; chemical binding; enzyme structure; genetic translation; membrane activity; nucleic acid sequence; protein transport; secretion

Our overall goal is to elucidate the molecular details of preprotein targeting to and translocation through biological membranes using the simple bacterium, Escherichia coli, as a facile genetic and biochemical system in this undertaking. Although rapid progress has been made in elucidating some of the basic features of E. coli protein secretion, most steps in this pathway are not understood in sufficient detail to construct a coherent biochemical scheme. Towards this goal we will focus on a central component of this system, SecA ATPase, which interacts with preproteins, the SecB chaperone, anionic phospholipids, integral membrane proteins SecY/SecE, as well as its own mRNA, and is the translocation ATPase required for preprotein insertion and passage across the inner membrane. Many of these interactions will be defined further, their molecular basis determined, their regulation by nucleotide binding and hydrolysis investigated, and their real purpose in the catalysis of protein secretion deciphered. In order to understand the function(s) of this complex ATPase, the distinct biochemical activities catalyzed by the two separate ATP-binding domains of SecA will be determine. The basis of SecA recognition of preproteins will be investigated employing a combined genetic and biochemical strategy to elucidate the structural features of the signal peptide and mature region of the preprotein that allow SecA recognition, and regions of SecA important in promoting these interactions will be determined. The basis of SecA's peripheral and integral associations with the inner membrane will be investigated further by identifying the SecA receptor and defining components that promote and regulate SecA's integral association with the membrane. A novel mapping technique employing cysteine-specific cleavage will be used to locate with high precision the ATP-binding, preprotein-binding, lipid- binding, and mRNA-binding sites on SecA's primary structure. A refined structural knowledge of SecA protein will be gained through collaborative studies to crystallize SecA protein or an important SecA substructure and determine its three dimensional structure at atomic resolution. Finally, the basis for SecA regulation and its coordination with the protein secretion status of the cell will be determine using genetic and biochemical approaches to locate the secretion-responsive element on geneX-secA mRNA, assess the importance of geneX translation termination and SecA protein binding in the function of this element, and identify any additional factors important in secA regulation. These studies should be of broad significance to understanding these basic problems in other protein secretion systems in normal and abnormal cells states as well as of practical importance in attempting to modify the export pathway to accommodate novel substrates.

我们的总体目标是阐明前蛋白的分子细节 使用生物膜靶向并易位 简单的细菌，大肠杆菌，作为一种简单的遗传和生化方法 此项事业的制度。 尽管在这方面取得了快速进展 阐明了大肠杆菌蛋白质分泌的一些基本特征，大多数 该途径中的步骤尚未得到足够详细的了解 构建连贯的生化方案。 为了这个目标，我们将重点 该系统的核心组件 SecA ATPase 与 前蛋白、SecB 伴侣、阴离子磷脂、整合膜 蛋白质 SecY/SecE 及其自身的 mRNA，是易位 前蛋白插入和穿过内膜所需的 ATP 酶 膜。 其中许多相互作用将被进一步定义，它们的 确定的分子基础，它们通过核苷酸结合的调节和 研究了水解，以及它们在催化中的真正用途 破译蛋白质分泌。 为了理解的功能 这种复杂的 ATP 酶，由 ATP 酶催化的独特生化活动 将确定 SecA 的两个独立的 ATP 结合域。 基础 SecA 对前蛋白的识别将采用 结合遗传和生化策略来阐明结构 信号肽和前蛋白成熟区域的特征 允许 SecA 认可，并且 SecA 区域在促进这些方面很重要 相互作用将被确定。 SecA外围设备的基础 将研究与内膜的整体关联 进一步鉴定 SecA 受体并定义其成分 促进和调节 SecA 与膜的整体结合。 一个 将使用采用半胱氨酸特异性切割的新型作图技术 高精度定位 ATP 结合、前蛋白结合、脂质 SecA 一级结构上的结合位点和 mRNA 结合位点。 一个精致的 SecA 蛋白的结构知识将通过合作获得 研究结晶 SecA 蛋白或重要的 SecA 子结构以及 以原子分辨率确定其三维结构。 最后， SecA 调节的基础及其与蛋白质的协调 细胞的分泌状态将通过遗传和 定位分泌反应元件的生化方法 geneX-secA mRNA，评估geneX翻译终止的重要性 与 SecA 蛋白结合该元件的功能，并鉴定 secA 调节中重要的任何其他因素。 这些研究 对于理解这些基本问题应该具有广泛的意义 正常和异常细胞中的其他蛋白质分泌系统状态为 以及在尝试修改出口方面具有实际重要性 适应新底物的途径。