Two homologous EF-G proteins from Pseudomonas aeruginosa exhibit distinct functions.

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 10(3) s(-1) M(-1) and 3.0 x 10(3) 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.

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.