Cloning and characterization of EF‐Tu and EF‐Ts from Bacillus subtilis

IntroductionElongation 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 of the ribosome. EF‐Ts acts by catalyzing the change of EF‐Tu from a GDP‐bound inactive state to a GTP‐bound active state. Much work has been done with elongation factors from Gram − bacteria but little work has been carried out to analyze the activity of these proteins from Gram + organisms. We describe here analysis of EF‐Tu and EF‐Ts from B. subtilis.ResultsAnalysis of both B. subtilis EF‐Tu and EF‐Ts showed they are 77% and 47% identical to their E. coli counterparts, respectively. Both B. subtilis EF‐Tu and EF‐Ts were cloned and over‐expressed in E. coli and purified to greater than 95% homogeneity. B. subtilis EF‐Tu was shown to be active when assayed for activity using the GDP binding assay. Kinetic parameters for the interaction of EF‐Tu with its substrate GDP were determined in the absence and presence of EF‐Ts. The dissociation constant (KGDP) for EF‐Tu•GDP was determined by forming the complex at 4 °C and diluting the complex 100 fold and measuring the amount of complex remaining at different times at various temperatures. EF‐Ts was shown to stimulated the exchange of GDP by EF‐Tu. EF‐Tu was also shown to be active in ternary complex formation as well as delivery of aminoacylated tRNA to the A site on B. subtilis ribosomes.ConclusionEF‐Tu and EF‐Ts identified in B. subtilis were cloned, expressed and purified and shown to be functional in assays suggesting that each is functional in protein synthesis.