MECHANISM OF PROTEIN LOCALIZATION IN ESCHERICHIA COLI

我们的总体目标是以大肠杆菌作为模型系统来阐明 ALL中发生的蛋白质定位的基本机制 活细胞。我们的长期目标是：(I)界定和 表征大肠杆菌蛋白质输出机制，(Ii)指定 对其各个组件的功能，(Iii)了解如何 这些功能相互关联，以产生一种有效的蛋白质 出口途径，以及(Iv)确定这一途径是如何调控的 并与其他基本的细胞过程相协调。在… 目前，已有9个出口机械部件 已鉴定，但尚未指定特定的生化功能 这些组件中的任何一个。这些活动的具体目标是 研究是为了进一步界定和阐明赛卡的作用。 在大肠杆菌中催化和调节蛋白质输出的基因产物。 使用突变分析和依赖于SecA的体外蛋白 移位系统，我们将定义移位 由SecA蛋白催化的活性，决定了哪些部分 编码这一活性的SecA多肽，并鉴定 SecA蛋白与Ins相互作用的出口相关成分 履行这一职能。我们将剖析Seca的共同调节。 与蛋白质出口熟练程度进一步结合使用 突变分析和体内、体外鉴定 SecA多肽的哪个部分编码它的自体 翻译抑制物活动，定义位置和结构 输出响应型翻译操作符，并阐明 对观察到的协同调节负责的生化机制。 由于蛋白质定位的机制是中心的 所有级别的生物重要性，从功能的角度来看 单个蛋白质分子在生物发生和维持中的作用 正常和异常的细胞状态，而且由于这种机制具有 在整个进化过程中都是保守的，这些对E.Coli的研究 应该具有广泛的意义。

Our overall goal is to use E.coli as a model system to elucidate the basic mechanisms of protein localization that occur in all living cells. Our long-term objective is (i) to define and characterize the E.coli protein export machinery, (ii) to assign functions to its individual components, (iii) to understand how these functions are interelated to produce an efficient protein export pathway, and (iv) to determine how this pathway is regulated and coordinated with other essential cellular processes. At present, 9 putative export machinery components have been identified, but no specific biochemical function has been assigned to any of these components. The specific objective of these studies is to further define and elucidate the role of the secA gene product in catalyzing and regulating protein export in E.coli. Using a mutational analysis and a SecA-dependent in vitro protein translocation system, we will define the translocation activity(ies) catalyzed by SecA protein, determine which portions of the SecA polypeptide encodes this activity, and identify the export-related components that SecA protein interacts with in fulfilling this function. We will dissect the coregulation of secA with protein export proficiency further using a combination of mutational analysis and in vivo and in vitro assays to characterize which portions of the SecA polypeptide encodes its autogenous translational repressor activity, define the location and structure of the export-responsive translational operator, and elucidate the biochemical mechanism responsible for the coregulation observed. Since the mechanisms of protein localization are of central biological importance at all levels, from the functioning of individual protein molecules to the biogenesis and maintenance of normal and abnormal cell states, and since such mechanisms have been conserved throughout evolution, these studies with E.coli should be of broad significance.