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Cloning and characterization of EF‐Tu and EF‐Ts from Pseudomonas aeruginosa
Author(s) -
Palmer Stephanie,
Rangle Edna,
Montalvo Albert,
Mugica Diego,
Bullard James
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.544.2
Subject(s) - ef tu , gtp' , chemistry , pseudomonas aeruginosa , elongation factor , biochemistry , ribosome , enzyme , biology , bacteria , gene , rna , genetics
Elongation factors (EF) facilitate many steps in the process of the biosynthesis of proteins. EF‐Tu in particular plays a central role in the GTP‐dependent placement of aminoacylated tRNA at the A site on the ribosome. EF‐Ts acts by catalyzing the change of EF‐Tu from a GDP‐bound inactive state to a GTP‐bound active state. Results Analysis of both P. aeruginosa EF‐Tu and EF‐Ts showed they are 84% and 55% identical to their E. coli counterparts, respectively. Both P. aeruginosa EF‐Tu and EF‐Ts were cloned and over‐expressed in E. coli and purified to greater than 95% homogeneity. EF‐Tu was shown to be active when assayed for activity using the GDP exchange assay. Kinetic parameters for the interaction of EF‐Tu with its substrate GDP were calculated to be: K M = 33 μM, V max = 0.125 μM/min, k cat = 1.65 × 10 −3 sec −1 . The specific activity of EF‐Tu was calculated to be 45 μM min −1 per mg of EF‐Tu. At a 1:20 ratio of EF‐Ts:EF‐Tu, EF‐Ts stimulated the exchange of GDP by EF‐Tu 10‐fold. Conclusion EF‐Tu and EF‐Ts identified in P. aeruginosa were cloned, expressed and purified and shown to be functional in GDP exchange assays suggesting that each is functional in protein synthesis.