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BioMed Research International
Volume 2013 (2013), Article ID 585748, 12 pages
http://dx.doi.org/10.1155/2013/585748
Research Article

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

Chemistry Department, SCIE. 3.320, The University of Texas-Pan American, 1201 W. University Drive, Edinburg, TX 78541, USA

Received 1 May 2013; Accepted 12 July 2013

Academic Editor: Leonid Medved

Copyright © 2013 Stephanie O. Palmer et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 = 33 μM, = 0.003 s−1, and the specificity constant was  s−1μM−1. In the presence of EF-Ts, these values were shifted to = 2 μM, = 0.005 s−1, and the specificity constant was  s−1μM−1. The equilibrium dissociation constants governing the binding of EF-Tu to GDP ( ) were 30–75 nM and to 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-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.