The Mechanism of Pausing and Restarting Translation in Bacteria

描述（由申请人提供）：延伸因子P（EF-P）在细菌蛋白质合成中起关键作用。EF-P的丧失导致多种表型，包括代谢应激、改变的抗生素抗性和毒力丧失。虽然EF-P在20世纪70年代被发现，但其生物学意义的第一个令人信服的解释出现在2013年初。当三个或更多连续的脯氨酸出现在新生肽中时，蛋白质合成暂停; EF-P与核糖体结合并恢复快速肽合成。我们的目标是实现一个分子水平的理解如何聚脯氨酸基序暂停翻译和暂停是如何解决EF-P。这些研究将揭示新生肽如何调节翻译速率，蛋白质折叠和基因调控的重要影响的现象。暂停的分子机制知之甚少。我们假设Pro改变了核糖体活性位点中关键核苷酸的几何形状，抑制了催化作用。在目标1中，我们将使用预稳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。我们将使用氨基酸类似物和rRNA突变来探测肽和活性位点的相互作用。我们还将研究EF-P如何抑制核糖体停顿。EF-P具有tRNA样结构，并与核糖体内的肽基-tRNA相互作用。位于肽基转移酶中心附近的Lys 34侧链被E-赖氨酰部分共价修饰，该部分是E.杆菌我们推测，修饰的侧链巧妙地重新排列了核糖体活性位点的几何形状，以恢复催化作用。在目标2中，我们将通过用一系列α-赖氨酸类似物化学修饰EF-P来测试α-赖氨酰部分的作用。值得注意的是，70%的细菌中缺乏负责β-赖氨酰化的酶YjeA和YjeK。我们将确定EF-P是如何在这些物种中的两个修改，并确定负责的酶。最后，我们的生物化学数据表明，EF-P在几个基序，而不仅仅是聚脯氨酸延伸的翻译暂停。在目标3中，我们将使用核糖体分析来定义EF-P活性的范围，核糖体分析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，我们将暂停与缺乏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 ß-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 ß-lysyl moiety by chemically modifying EF-P with a series of ß-lysine analogs. Notably, the enzymes responsible for ß-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.