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 mu M, k(cat)(obs) = 0.003 s(-1), and the specificity constant k(cat)(obs)/K-M was 0.1 x 10(-3) s(-1) mu M-1. In the presence of EF-Ts, these values were shifted to K-M = 2 mu M, k(cat)(obs) = 0.005 s(-1), and the specificity constant k(cat)(obs)/K-M was 2.5 x 10(-3) s(-1) mu M-1. The equilibrium dissociation constants governing the binding of EF-Tu to GDP (K-GDP) were 30-75nM 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.