Regulation of Protein Synthesis in Bacteria by Ser/Thr Phosphorylation

Ser/Thr 磷酸化调节细菌蛋白质合成

• 批准号: 9264543
• 负责人: JONATHAN DWORKIN
• 金额: $ 37.85万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-15 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9264543
• 关键词: Affect; Amino Acyl Transfer RNA; Antibiotics; Attenuated; Bacteria; Biochemical; Cells; Dominant-Negative Mutation; Down-Regulation; Enzymes; Escherichia coli; Gram-Positive Bacteria; Growth; In Vitro; Mediating; Messenger RNA; Metabolic; Methods; Modification; Nature; Nutrient; Peptide Elongation Factor G; Peptide Elongation Factor Tu; Phase; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Phylogenetic Analysis; Physiological; Play; Post-Translational Protein Processing; Process; Production; Protein Biosynthesis; Protein Dephosphorylation; Proteins; Recycling; Regulation; Resources; Ribosomes; Role; Transcriptional Regulation; Translations; Work; biophysical techniques; experimental study; in vivo; novel; public health relevance; rapid growth; response; translation factor

 DESCRIPTION (provided by applicant): Protein synthesis consumes most of the cell's energy during rapid growth. As growth slows during nutrient limitation, the cell reduces protein synthesis. In order for the cell to quickly resume growth when nutrients become available, the down regulation of protein synthesis must be reversible. One method the cell employs to regulate protein synthesis is by transcriptional regulation of the protein synthesis machinery. However, this method would require de novo ribosome production when growth resumes. In contrast, relatively stable post-translational modifications like Ser/Thr phosphorylation could provide rapid and reversible control of protein synthesis by modifying existing proteins. Most bacteria contain Ser/Thr kinases and phosphatases and among the targets of these enzymes are the essential translation factors Elongation Factor Tu (EF-Tu) and Elongation Factor G (EF-G). Thus, Ser/Thr phosphorylation may regulate protein synthesis in bacteria by modulating the activities of EF-Tu and EF-G. Here, we investigate this hypothesis and identify specific Ser/Thr kinases and phosphatases that mediate the reversible phosphorylation of EF- Tu and EF-G in B. subtilis and E. coli. We will characterize how these modifications affect the function of EF-Tu and EF-G in vitro using biochemical and biophysical techniques. We also investigate the in vivo consequences of these modifications, with particular focus on sporulation in B. subtilis and stationary phase in E. coli, both physiological situations where protein synthesis is attenuated in response to nutrient limitation. This work will provide a new framework for understanding how protein synthesis is regulated in bacteria by reversible Ser/Thr phosphorylation. Our finding that Ser/Thr phosphorylation of two translation factors inhibits their activity and results in a dominan negative inhibition of elongation suggests how a process dependent on very abundant proteins can be sensitively regulated. In addition, the reversible nature of this mechanism allows cells both to enter and to exit metabolic quiescence in response to changes in nutrient availability. Finally, our characterization of these mechanisms in both E. coli and B. subtilis suggest that they are phylogenetically conserved.

 描述（由申请人提供）：蛋白质合成在快速生长过程中消耗了细胞的大部分能量。当营养限制期间生长减慢时，细胞会减少蛋白质合成。为了使细胞在营养物质可用时快速恢复生长，蛋白质合成的下调必须是可逆的。细胞调节蛋白质合成的一种方法是通过蛋白质合成机制的转录调节。然而，当生长恢复时，这种方法需要从头开始生产核糖体。相比之下，相对稳定的翻译后修饰（例如 Ser/Thr 磷酸化）可以通过修饰现有蛋白质来提供对蛋白质合成的快速且可逆的控制。大多数细菌含有 Ser/Thr 激酶和磷酸酶，这些酶的靶标包括重要的翻译因子延伸因子 Tu (EF-Tu) 和延伸因子 G (EF-G)。因此，Ser/Thr 磷酸化可能通过调节 EF-Tu 和 EF-G 的活性来调节细菌中的蛋白质合成。在这里，我们研究了这一假设，并鉴定了介导枯草芽孢杆菌和大肠杆菌中 EF-Tu 和 EF-G 可逆磷酸化的特定 Ser/Thr 激酶和磷酸酶。我们将使用生物化学和生物物理技术描述这些修饰如何影响 EF-Tu 和 EF-G 的体外功能。我们还研究了这些修饰的体内后果，特别关注枯草芽孢杆菌中的孢子形成和大肠杆菌中的静止期，这两种生理情况下蛋白质合成在 对营养限制的反应。这项工作将为理解细菌中蛋白质合成如何通过可逆的丝氨酸/苏氨酸磷酸化调节提供一个新的框架。我们发现两个翻译因子的 Ser/Thr 磷酸化会抑制它们的活性并导致延伸的显性负抑制，这表明如何敏感地调节依赖于非常丰富的蛋白质的过程。此外，这种机制的可逆性使细胞能够响应营养物质可用性的变化而进入和退出代谢静止状态。最后，我们对大肠杆菌和枯草芽孢杆菌中这些机制的表征表明它们在系统发育上是保守的。