Cloning and Characterization of EF-Tu and EF-Ts from Pseudomonas aeruginosa. (5th August 2013)

• Record Type: Journal Article
• Authors: Palmer, Stephanie O.; Rangel, Edna Y.; Montalvo, Alberto E.; Tran, Alexis T.; Ferguson, Kate C.; Bullard, James M.
• Other Names: Medved Leonid Academic Editor.
• Is Part Of: BioMed research international. Volume 2013(2013)
• Publication Date: 2013-08-05
• Subjects: Medicine -- Periodicals; Biology -- Periodicals; Biotechnology -- Periodicals; Life sciences -- Periodicals; 610.5
• DOI: 10.1155/2013/585748
• Languages: English
• ISSNs: 2314-6133

Abstract

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