Regulation of Protein Synthesis in Bacteria by Ser/Thr Phosphorylation

 描述（由申请人提供）：蛋白质合成在快速生长期间消耗细胞的大部分能量。随着生长在营养限制期间减慢，细胞减少蛋白质合成。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。细胞用来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，这种方法需要从头产生核糖体。相比之下，相对稳定的翻译后修饰如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和可逆的控制。大多数细菌含有Ser/Thr激酶和磷酸酶，并且这些酶的靶标是必需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。在此，我们研究了这一假设，并鉴定了介导B中EF-Tu和EF-G可逆磷酸化的特异性Ser/Thr激酶和磷酸酶。枯草芽孢杆菌和E.杆菌我们将使用生物化学和生物物理技术来表征这些修饰如何在体外影响EF-Tu和EF-G的功能。我们还研究了这些修饰的体内后果，特别关注B中的孢子形成。subtilis和E.大肠杆菌，这两种生理情况下，蛋白质合成减弱， 对营养限制的反应。这项工作将提供一个新的框架，了解蛋白质合成是如何在细菌中调节可逆的丝氨酸/苏氨酸磷酸化。我们的发现，Ser/Thr磷酸化的两个翻译因子抑制其活性，并在一个显性负抑制延伸的结果表明，如何依赖于非常丰富的蛋白质的过程可以灵敏地调节。此外，这种机制的可逆性质允许细胞响应于营养可用性的变化而进入和退出代谢静止。最后，我们的表征这些机制在E。coli和B.枯草芽孢杆菌的基因组序列表明它们在遗传学上是保守的。

 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.