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

Abstract EN

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 KM = 33 μM, kcatobs = 0.003 s−1, and the specificity constant kcatobs/KM was 0.1×10-3 s−1 μM−1.

In the presence of EF-Ts, these values were shifted to KM = 2 μM, kcatobs = 0.005 s−1, and the specificity constant kcatobs/KM was 2.5×10-3 s−1 μM−1.

The equilibrium dissociation constants governing the binding of EF-Tu to GDP (KGDP) were 30–75 nM and to GTP (KGTP) 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.