Cloning and characterization of EF-Tu and EF-Ts from Pseudomonas aeruginosa.

ABSTRACT: 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 ?M, k cat (obs) = 0.003 s(-1), and the specificity constant k cat (obs)/K M was 0.1 × 10(-3) s(-1) ?M(-1). In the presence of EF-Ts, these values were shifted to K M = 2 ?M, k cat (obs) = 0.005 s(-1), and the specificity constant k(cat)(obs)/K M was 2.5 × 10(-3) s(-1) ?M(-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.

SUBMITTER: Palmer SO 

PROVIDER: S-EPMC3747624 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature