Cloning and characterization of two EF‐G proteins from Pseudomonas aeruginosa

Elongation factors (EF) facilitate many steps in the translation process. EF‐G in particular plays a central role in catalyzing GTP‐dependent translocation and is also involved in GTP‐dependent termination of protein synthesis. Genetic analysis of the P. aeruginosa genome revealed the presence of two forms of EF‐G: EF‐G1 and EF‐G2. Results Analysis of both forms of P. aeruginosa EF‐G showed they are 84% identical and both are 67% identical to E. coli EF‐G. Each form of P. aeruginosa EF‐G was cloned and over‐expressed in E. coli and purified to greater than 98% homogeneity. Both of the enzymes were active in ribosome dependent‐GTPase assays. EF‐G1 converted GTP to GDP and P i with a specific activity of 232 μM min −1 mg while EF‐G2 produced P i with a specific activity of 2214 μM min −1 mg. The 10‐fold increased activity of EF‐G2 suggests a role in the elongation phase of translation. Neither form of EF‐G was active in the absence of ribosomes. The activity of both forms of EF‐G was affected by the addition of fusidic acid to the reaction. Conclusion Two forms of EF‐G identified in P. aeruginosa are both similar to EF‐G from E. coli and both forms were shown to be functional in ribosome‐dependent GTPase activity suggesting that each is functional in protein synthesis. Research was supported by the South Texas Border Health Disparities Center (grant H75DP001812 from the Centers for Disease Control and Prevention).