Cloning and Characterization of EF-Tu and EF-Ts from Pseudomonas aeruginosa

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-tRNA(Phe) 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.