Regulation of Protein Synthesis in Bacteria by Ser/Thr Phosphorylation

Columbia University Health Sciences, New York NY

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Abstract

? 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.