Regulation of Protein Synthesis in Bacteria by Ser/Thr Phosphorylation

 描述(申请人提供)：蛋白质合成在快速生长过程中消耗细胞的大部分能量。在营养限制期间，随着生长速度减慢，细胞蛋白质合成减少。为了让细胞在获得营养后迅速恢复生长，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，这种方法需要从头开始生产核糖体。相反，相对稳定的翻译后修饰，如Ser/Thr磷酸化，可以通过修改现有的蛋白质来快速和可逆地控制蛋白质的合成。大多数细菌都含有丝氨酸/苏氨酸蛋白激酶和磷酸酶，在这些酶的靶标中，有两个重要的翻译因子：伸长因子Tu(EF-Tu)和伸长因子G(EF-G)。因此，丝氨酸/苏氨酸的磷酸化可能通过调节EF-Tu和EF-G的活性来调节细菌蛋白质的合成。在这里，我们研究了这一假说，并确定了在枯草杆菌和大肠杆菌中介导EF-Tu和EF-G可逆磷酸化的特定Ser/Thr激酶和磷酸酶。我们将使用生化和生物物理技术在体外表征这些修饰如何影响EF-Tu和EF-G的功能。我们还研究了这些修饰在体内的后果，特别关注枯草杆菌中的产孢子和大肠杆菌中的静止相，这两种生理情况下蛋白质合成都会减弱。 对营养限制的反应。这项工作将为理解细菌中蛋白质合成是如何通过可逆的Ser/Thr磷酸化调节的提供了一个新的框架。我们的发现是，两个翻译因子的Ser/Thr磷酸化抑制了它们的活性，并导致了对伸长的显性负抑制，这表明依赖于非常丰富的蛋白质的过程可以被敏感地调节。此外，这种机制的可逆性允许细胞进入和退出代谢静止状态，以响应养分供应的变化。最后，我们在大肠杆菌和枯草杆菌中对这些机制的描述表明，它们在系统发育上是保守的。

 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.