The Mechanism of Pausing and Restarting Translation in Bacteria

描述(申请人提供)：延伸因子P(EF-P)在细菌的蛋白质合成中起着关键作用。EF-P的丢失会导致多种表型，包括代谢应激、抗生素耐药性的改变和毒力的丧失。虽然EF-P是在20世纪70年代的S被发现的，但对其生物学意义的第一次令人信服的解释出现在2013年初。当新生多肽中出现三个或三个以上连续的脯氨酸时，蛋白质合成暂停；EF-P与核糖体结合，恢复快速的多肽合成。我们的目标是在分子水平上理解多聚脯氨酸基序如何暂停翻译，以及EF-P如何解决暂停。这些研究将阐明这种新生多肽如何调节翻译速率，这一现象对蛋白质折叠和基因调控具有重要意义。停顿的分子机制还知之甚少。我们假设Pro改变了核糖体活性部位的关键核苷酸的几何形状，从而抑制了催化作用。在目标1中，我们将使用预稳定状态动力学方法来测试这一假设，以隔离导致暂停的速率缺陷。我们将利用氨基酸类似物和rRNA突变来探索多肽与活性部位的相互作用。我们还将研究EF-P如何减轻核糖体停顿。EF-P具有类似tRNA的结构，并与核糖体内的多肽-tRNA相互作用。Lys34侧链位于肽基转移酶中心附近，它被？-赖氨酰部分共价修饰，这是大肠杆菌EF-P功能所必需的。我们假设修饰后的侧链 微妙地重新排列核糖体活性部位的几何结构，以恢复催化作用。在目标2中，我们将 通过用一系列赖氨酸类似物对EF-P进行化学修饰来测试？-赖氨酸部分的作用。值得注意的是，负责裂解的酶YjeA和YjeK在70%的细菌中缺乏。我们将确定EF-P在其中两个物种中是如何被修饰的，并确定相关的酶。最后，我们的生化数据表明，EF-P减少了几个基序的翻译停顿，而不仅仅是多聚脯氨酸延伸。在目标3中，我们将使用核糖体图谱来定义EF-P活性的范围，核糖体图谱是一种在活细胞中全局监测翻译的方法。通过监测翻译和mRNA表达的变化，我们将把暂停与在缺乏EF-P的细胞中观察到的多效性表型联系起来。

DESCRIPTION (provided by applicant): Elongation factor P (EF-P) plays a critical role in protein synthesis in bacteria. Loss of EF- P leads to a variety of phenotypes, including metabolic stress, altered antibiotic resistance, and loss of virulence. Although EF-P was discovered in the 1970's, the first compelling explanation of its biological significance appeared in early 2013. Protein synthesis pauses when three or more consecutive prolines occur in the nascent peptide; EF-P binds to the ribosome and restores rapid peptide synthesis. Our goal is to achieve a molecular-level understanding of how polyproline motifs pause translation and how pauses are resolved by EF-P. These studies will shed light on how the nascent peptide modulates translational rates, a phenomenon with important implications for protein folding and gene regulation. The molecular mechanism of pausing is poorly understood. We hypothesize that Pro alters the geometry of key nucleotides in the ribosomal active site, inhibiting catalysis. In Aim 1 we will test this hypothesis using pre- steady state kinetic methods to isolate rate defects that lead to pausing. We will probe the interaction of the peptide and active site using amino acid analogs and rRNA mutations. We will also investigate how EF-P alleviates ribosome pausing. EF-P has a tRNA-like structure and interacts with peptidyl-tRNA within the ribosome. The Lys34 side chain, positioned near the peptidyl-transferase center, is covalently modified with a ?-lysyl moiety that is essential for EF- P function in E. coli. We hypothesize that the modified side chain subtly rearranges the geometry of the ribosomal active site to restore catalysis. In Aim 2, we will test the role of the ?-lysyl moiety by chemically modifying EF-P with a series of ¿-lysine analogs. Notably, the enzymes responsible for ?-lysylation, YjeA and YjeK, are lacking in 70% of bacteria. We will determine how EF-P is modified in two of these species and identify the enzymes responsible. Finally, our biochemical data suggest that EF-P alleviates translational pausing at several motifs, not just polyproline stretches. In Aim 3, we will define the scope of EF-P activity using ribosome profiling, a method for monitoring translation globally in living cell. By monitoring changes in translation and mRNA expression, we will link pausing to the pleiotropic phenotypes observed in cells lacking EF-P.