Studies on phosphorylation of elongation factor Tu in escherichia coli

Abstract

Protein synthesis is a vital process for living organisms. Synthesis of proteins are important for cell survival and reproduction. Protein synthesis takes place on ribosomes in all prokaryotic and eukaryotic cells. Amino acids of the proteins are carried to the ribosome by transfer RNA molecules in a ternary complex as EF-Tu.GTP.aminoacyl-tRNA. EF-Tu is the most abundant protein in bacterial cells, reaching about 10% in logaritmically growing cells. Due to its vital function in translation, EF-Tu is target to many translational antibiotics and also to many post-translational modifications. EF-Tu is phosphorylated in vivo in both prokaryotic and eukaryotic cells. Phosphorylation of EF-Tu at position T382 abolishes its ability to form EF-Tu.GTP.aminoacyl-tRNA ternary complex, indicating that protein synthesis can be regulated reversibly by EF-Tu phosphorylation of T382. Therefore, characterization of the kinase, specific for T382 phosphorylation, is the first step in order to define the members of the signaling pathway leading to translational arrest. Phosphorylation of EF-Tu at T382 has the capability of rapid translational arrest. This mechanism is the possible way for the bacteria to enter into the dormant state during bacterial persistence. Therefore, finding the kinase in the pathway inhibiting translation can open away to design new antibiotics in order to eradicate bacterial persistence. Even clarifying the mechanisms of bacterial persistence can give an insight for the treatment of cancer because cancer cells are also slow-growing and drug-tolerant; and they revert back to malignant phenotype and rapid growing state similar to persistent bacteria after treatment is removed. In the framework of this thesis, candidate genes were determined by literature searching and by using bioinformatics tools. BlastP and PSI-Balst were used in order to detect amino acid sequence similarity. FATCAT programme was used to detect structural similarity. hipA gene was determined as the first candidate gene by literature searching. yegI gene and argK genes were determined as other candidates by bioinformatics searches. These genes were cloned and purified in order to identify the kinase that phosphorylates EF-Tu at T382. Phosphorylation assay was performed with purified kinases and pure EF-Tu. Phosphorylation of EF-Tu at T382 was detected by anti-phosphothreonine antibody. Mutants of EF-Tu at T382 were used as controls for T382 specificity. It was detected that none of these proteins are able to phosphorylate EF-Tu at T382.