Genes encoding two proteins corresponding to elongation factor G (EF-G) were cloned from Pseudomonas aeruginosa. The proteins encoded by these genes are both members of the EFG I subfamily. The gene encoding one of the forms of EF-G is located in the str operon and the resulting protein is referred to as EF-G1A while the gene encoding the other form of EF-G is located in another part of the genome and the resulting protein is referred to as EF-G1B. These proteins were expressed and purified to 98% homogeneity. Sequence analysis indicated the two proteins are 90/84% similar/identical. In other organisms containing multiple forms of EF-G a lower degree of similarity is seen. When assayed in a poly(U)-directed poly-phenylalanine translation system, EF-G1B was 75-fold more active than EF-G1A. EF-G1A pre-incubate with ribosomes in the presence of the ribosome recycling factor (RRF) decreased polymerization of poly-phenylalanine upon addition of EF-G1B in poly(U)-directed translation suggesting a role for EF-G1A in uncoupling of the ribosome into its constituent subunits. Both forms of P. aeruginosa EF-G were active in ribosome dependent GTPase activity. The kinetic parameters (K M) for the interaction of EF-G1A and EF-G1B with GTP were 85 and 70 μM, respectively. However, EF-G1B exhibited a 5-fold greater turnover number (observed k cat) for the hydrolysis of GTP than EF-G1A; 0.2 s-1 vs. 0.04 s-1. These values resulted in specificity constants (k cat obs/K M) for EF-G1A and EF-G1B of 0.5 x 103 s-1 M-1 and 3.0 x 103 s-1 M-1, respectively. The antibiotic fusidic acid (FA) completely inhibited poly(U)-dependent protein synthesis containing P. aeruginosa EF-G1B, but the same protein synthesis system containing EF-G1A was not affected. Likewise, the activity of EF-G1B in ribosome dependent GTPase assays was completely inhibited by FA, while the activity of EF-G1A was not affected.

We have cloned genes encoding elongation factors EF-Tu and EF-Ts from Pseudomonas aeruginosa and expressed and purified the proteins to greater than 95% homogeneity. Sequence analysis indicated that P. aeruginosa EF-Tu and EF-Ts are 84% and 55% identical to E. coli counterparts, respectively. P. aeruginosa EF-Tu was active when assayed in GDP exchange assays. Kinetic parameters for the interaction of EF-Tu with GDP in the absence of EF-Ts were observed to be K M = 33 μM, k cat obs = 0.003 s−1, and the specificity constant k cat obs/K M was 0.1 × 10−3 s−1  μM−1. In the presence of EF-Ts, these values were shifted to K M = 2 μM, k cat obs = 0.005 s−1, and the specificity constant k cat obs/K M was 2.5 × 10−3 s−1  μM−1. The equilibrium dissociation constants governing the binding of EF-Tu to GDP (K GDP) were 30–75 nM and to GTP (K GTP) were 125–200 nM. EF-Ts stimulated the exchange of GDP by EF-Tu 10-fold. P. aeruginosa EF-Tu was active in forming a ternary complex with GTP and aminoacylated tRNA and was functional in poly(U)-dependent binding of Phe-tRNAPhe at the A-site of P. aeruginosa ribosomes. P. aeruginosa EF-Tu was active in poly(U)-programmed polyphenylalanine protein synthesis system composed of all P. aeruginosa components.

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IntroductionElongation factors (EF) facilitate many steps in the process of the biosynthesis of proteins. EF‐Tu in particular plays a central role in the GTP‐dependent placement of aminoacylated tRNA at the A site of the ribosome. EF‐Ts acts by catalyzing the change of EF‐Tu from a GDP‐bound inactive state to a GTP‐bound active state. We present here analysis of the kinetic parameters of EF‐Tu and EF‐Ts.ResultsKinetic parameters for the interaction of EF‐Tu with its substrate GDP in the absence of EF‐Ts were observed to be: KM = 33 mM, kcat = 0.003 sec−1 and the specificity constant kcat/KM was then calculated as 0.1 × 10−3 sec−1 uM−1. In the presence of EF‐Ts, these values were shifted to: KM = 2 mM, kcat = 0.005 sec−1 and the specificity constant kcat/KM was then calculated as 2.5 × 10−3 sec−1 uM−1. We determined the equilibrium dissociation constant (KGDP) to be in the range of 100–160 nM in the absence of EF‐Ts at various temperatures. The presence of EFTs stimulated the exchange of GDP by EF‐Tu by up to 10‐fold. P. aeruginosa EF‐Tu was also shown to be active in forming a ternary complex with GTP and aminoacylated tRNA and was functional in polyU dependent placement of Phe‐tRNAPhe at the A site of the P. aeruginosa ribosome.ConclusionEF‐Tu and EF‐Ts identified in P. aeruginosa were cloned, expressed and purified and shown to be functional in assays suggesting that each is functional in protein synthesis. Research was supported by NIH grant 1SC3GM098173–01A1.