The Mechanism of Pausing and Restarting Translation in Bacteria

细菌暂停和重新启动翻译的机制

• 批准号: 8876739
• 负责人: Allen Rowdon Buskirk
• 金额: $ 30.78万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-20 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8876739
• 关键词: Active Sites; Affect; Amino Acids; Antibiotic Resistance; Bacteria; Binding; Biochemical; Biological; Catalysis; Cells; Data; Data Analyses; Defect; Enzymes; Escherichia coli; Eukaryota; Gene Expression; Gene Expression Regulation; Geometry; Goals; In Vitro; Kinetics; Label; Lead; Length; Life; Light; Link; Measures; Mediating; Messenger RNA; Metabolic stress; Methods; Modification; Molecular; Monitor; Mutation; Nucleotides; Organism; Orthologous Gene; Peptide Leader Sequences; Peptide Synthesis; Peptides; Peptidyltransferase; Phenotype; Play; Positioning Attribute; Proline; Protein Biosynthesis; Proteins; Ribosomal RNA; Ribosomes; Role; Salmonella; Series; Side; Site; Stretching; Structure; System; Techniques; Testing; Transfer RNA; Translations; Virulence; analog; antibiotic design; density; factor EF-P; functional group; genome-wide; insight; lysine analog; mRNA Expression; mutant; novel; peptidyl-tRNA; polyproline; protein folding; public health relevance; tool

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.

描述（应用程序提供）：伸长因子P（EF-P）在细菌中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，抗生素耐药性改变和病毒丧失。尽管EF-P是在1970年代发现的，但对其生物学意义的第一个引人注目的解释出现在2013年初。蛋白质合成暂停了三个或更多连续的脯氨酸EF-P与核糖体结合并恢复快速肽合成。我们的目标是实现分子级的理解，以了解多发性基序如何停顿翻译以及如何通过EF-P解决暂停。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节具有重要意义。暂停的分子机制知之甚少。我们假设核糖体活性位点中关键核苷酸的几何形状，从而抑制催化。在AIM 1中，我们将使用稳态的状态动力学方法检验该假设，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突变探测胡椒和活性位点的相互作用。我们还将研究EF-P如何减轻核糖体的暂停。 EF-P具有tRNA状结构，并与核糖体内的肽基-TRNA相互作用。位于Petidyl-转移酶中心附近的Lys34侧链，用EF-P功能在大肠杆菌中必不可少的β-溶酶部分进行了共价修饰。我们假设修改后的侧链巧妙地重新排列了核糖体活动部位的几何形状以恢复催化。在AIM 2中，我们将通过用一系列β-赖氨酸类似物对EF-P进行化学修饰EF-P来测试β-溶部分部分的作用。值得注意的是，在70％的细菌中缺乏负责β-溶解酶的酶。我们将确定如何在其中两个物种中修改EF-P并确定负责的酶。最后，我们的生化数据表明，EF-P可以减轻几个基序的平移暂停，而不仅仅是多产延伸。在AIM 3中，我们将使用核糖体分析定义EF-P活性的范围，核糖体分析是一种在活细胞中全球监测翻译的方法。通过监测翻译和mRNA表达的变化，我们将暂停与缺乏EF-P细胞中观察到的多效性表型联系起来。