Cloning and Characterization of EF-Tu and EF-Ts from Pseudomonas aeruginosa
Published Date:Aug 05 2013
Source:Biomed Res Int. 2013; 2013.
Keywords:Amino Acid Sequence
Molecular Sequence Data
Peptide Elongation Factors
Peptide Elongation Factor Tu
Sequence Analysis, Protein
Sequence Homology, Amino Acid
Pubmed Central ID:PMC3747624
Funding:1SC3GM098173-01A1/GM/NIGMS NIH HHS/United States
75DP001812/DP/NCCDPHP CDC HHS/United States
SC3 GM098173/GM/NIGMS NIH HHS/United States
Description: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.
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