Abstract:
Elongation factor Tu (EF-Tu) plays a crucial role of carrying aminoacylated-tRNA molecules to the ribosome during the elongation phase of protein synthesis. Being a guanine nucleotide binding protein, EF-Tu is inactive when in complex with GDP whereas binding of GTP switches the protein to its active state in which EF-Tu forms a ternary complex with an aminoacylated-tRNA alongside GTP. Moreover, a critical phosphorylation of the conserved threonine in the 382th position of Escherichia coli EF-Tu abolishes the ability of EF-Tu to form a ternary complex. Although this phosphoryla-tion taking place in vivo and its effect on EF-Tu function has been revealed long ago, the responsible kinase for this phosphorylation remained unknown. In this study, the critical threonine is substituted by alanine, aspartic acid or serine in order to further analyze the role of this residue in EF-Tu function. None of the mutations have an effect on GDP or GTP binding capability of EF Tu. However, only the serine mutant retains the ability to bind aminoacylated-tRNA whereas the other two fail either to make or to preserve the bond as successful as the wild type. The T382 mutants are also used as negative or neu-tral controls in the process of searching for the protein kinase responsible for the EF-Tu phosphorylation. A eukaryotic like serine/threonine kinase in Bacillus subtilis, PrkC, was shown to be specifically phosphorylating the critical threonine residue in vitro in a recent study, which in turn led our study to a search of PrkC homolog in Escherichia coli. A recently identified protein kinase, RdoA, showed similarities to PrkC in its amino acid sequence and is chosen as the candidate protein kinase. Eventually, RdoA was cloned and expressed as a recombinant protein and the purified protein was used in in vitro experiments. EF-Tu is shown to be phosphorylated in its T382 residue by RdoA. However, the questions whether RdoA is the specific kinase that phosphorylates EF-Tu in vivo remains unanswered.
