The Mechanism of Pausing and Restarting Translation in Bacteria

描述（由申请人提供）：伸长因子P （EF-P）在细菌的蛋白质合成中起关键作用。EF- P的缺失导致多种表型，包括代谢应激、抗生素耐药性改变和毒力丧失。虽然EF-P早在20世纪70年代就被发现，但对其生物学意义的第一个令人信服的解释出现在2013年初。当新生肽中出现三个或更多连续的脯氨酸时，蛋白质合成暂停；EF-P与核糖体结合，恢复快速肽合成。我们的目标是在分子水平上理解聚脯氨酸基序是如何暂停翻译的，以及EF-P是如何解决暂停的。这些研究将阐明新生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。暂停的分子机制尚不清楚。我们假设Pro改变了核糖体活性部位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检验这一假设，以分离导致暂停的速率缺陷。我们将利用氨基酸类似物和rRNA突变来探索肽和活性位点的相互作用。我们还将研究EF-P如何缓解核糖体暂停。EF-P具有trna样结构，并与核糖体内的肽基trna相互作用。Lys34侧链位于肽基转移酶中心附近，与大肠杆菌中EF- P功能所必需的ß-赖氨酸片段共价修饰。我们假设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Aim 2中，我们将通过一系列ß-赖氨酸类似物对EF-P进行化学修饰来测试ß-赖氨酸片段的作用。值得注意的是，70%的细菌缺乏负责ß-赖基化的酶YjeA和YjeK。我们将确定EF-P如何在其中两个物种中被修饰，并确定负责的酶。最后，我们的生化数据表明，EF-P缓解了几个基序上的翻译停顿，而不仅仅是脯氨酸延伸。在Aim 3中，我们将使用核糖体分析（一种在活细胞中监测翻译全局的方法）来定义EF-P活性的范围。通过监测翻译和mRNA表达的变化，我们将把暂停与缺乏EF-P的细胞中观察到的多效表型联系起来。

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.