Functional role of the conserved amino acids Cysteine 81, Arginine 279, Glycine 280 and Arginine 283 in elongation factor Tu from Escherichia coli

During protein synthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of mRNA-programmed ribosomes in a GTP-dependent manner. To enable future studies on the functional and structural requirement of EF-Tu’s function, a Cysteine-free variant of EF-Tu was constructed suitable for subsequent labelling of the protein and use in kinetic studies. Here, the kinetic properties of three Cysteine-less EF-Tu variants are reported, demonstrating that only the variant with the Alanine substitution in position 81 retains wild-type activity with respect to the interaction with guanine nucleotides, aa-tRNA and the ribosome. To explore a possible tRNA independent pathway for the GTPase activation signal, three residues in domain II of EF-Tu (Arginine 279, Glycine 280, Arginine 283) were mutated; the activity of EF-Tu variants were analyzed. Results suggest that these residues are indeed required for efficient ribosome-dependent stimulation of the GTPase activity of EF-Tu.

Author

• Mo, Fan

• Aminoacyl-tRNA
• Guanosine triphosphatase
• Proteins -- Synthesis
• Binding sites (Biochemistry)
• Genetic translation
• Escherichia coli
• Dissertations, Academic

Identifier

hdl:10133/3107

OAI identifier oai:identifier

oai:opus.uleth.ca:10133/3107