Bacterial protein tagging, degradation & ribosome rescue

描述(申请人提供)：本研究计划的目标是了解大肠杆菌的SsrA(TmRNA)系统，探索其在核糖体拯救、蛋白质标记和其他细胞过程中的作用，并确定细菌和带有SSrA标记或其他降解信号的噬菌体蛋白如何被细菌蛋白酶识别和降解。我们将探索导致SsrA标签的mRNA和/或蛋白质决定因素，研究全长蛋白质标签的生物学功能，并确定哪些大分子因素与tmRNA周期的不同部分与SsrA RNA相关。我们还将研究ClpXP和SspB的结构、功能和底物结合特异性。ClpXP是降解ssrA标记蛋白的主要蛋白酶，SspB是一种与ClpXP合作促进ssrA标记蛋白降解的调节因子。抑制子和其他重要的调节因素是ClpXP降解的常见目标。了解SsrA的功能、核糖体拯救和蛋白质降解是分子和结构生物学基础研究的关键目标，在医学、生物技术以及设计新的蛋白质和调节电路方面具有潜在的应用前景。例如，SsrA系统对于细菌病原体的传染性是必需的，并允许细菌耐受更高剂量的抑制蛋白质合成的抗生素。对SsrA标记位置的分析揭示了核糖体受损的位置，提供了阻碍或阻碍蛋白质生物合成的分子事件的独特一瞥。这样的信息可以改善重组蛋白的表达。ClpX既是ClpXP蛋白水解酶的调节亚单位，也是AAA+家族的拆卸伴侣。对ClpX底物识别的详细了解可以使设计的酶具有改变的特异性，用作发现研究的工具。ClpX、ClpXP和SspB也是理解分子机制的模型，这些机制从细菌到人类都是保守的，并在从蛋白质降解到膜融合的各种过程中被所有细胞适应。

DESCRIPTION (provided by applicant): The objective of this research program is to understand the SsrA (tmRNA) system of E. coli, to probe its roles in ribosome rescue, protein tagging, and other cellular processes, and to determine how bacterial and phage proteins bearing ssrA tags or other degradation signals are recognized and degraded by bacterial proteases. We will probe the mRNA and/or protein determinants that induce SsrA tagging, study the biological function of full-length protein tagging, and determine which macromolecular factors associate with SsrA RNA during different parts of the tmRNA cycle. We will also study the structure, function, and substrate-binding specificity of ClpXP, the major protease that degrades ssrA-tagged proteins, and SspB, a modulatory factor that collaborates with ClpXP to enhance degradation of ssrA-tagged proteins. Repressors and other important regulatory factors are frequent targets of ClpXP degradation. Understanding SsrA function, ribosome rescue, and protein degradation are key goals of basic research in molecular and structural biology, with potential applications in medicine, biotechnology, and the design of novel proteins and regulatory circuits. For example, the SsrA system is required for the infectivity of bacterial pathogens and allows bacteria to withstand higher doses of antibiotics that inhibit protein synthesis. Analysis of the sites of SsrA tagging reveals locations of ribosome distress, providing a unique glimpse of the molecular events that hinder or impede protein biosynthesis. Such information could permit improved expression of recombinant proteins. ClpX serves both as the regulatory subunit of the ClpXP protease and as an AAA+ family disassembly chaperone. A detailed knowledge of ClpX-substrate recognition could allow the design of enzymes with altered specificity for use as tools in discovery research. ClpX, ClpXP, and SspB also serve as models in which to understand molecular mechanisms that have been conserved from bacteria to humans and adapted by all cells in processes ranging from protein degradation to membrane fusion.