Chemical and physical studies on the structure of Escherichia coli elongation factor G.

Elongation Factor G (EF-G) from Escherichia coli was purified to homogenity by a previously published method (Rohrbach, M. S., Dempsey, M. E., and Bodley, J. W. (1974) J. Biol. Chem. 249, 5094). The protein is composed of a single polypeptide chain of molecular weight 74,000 under native conditions and 71,000 under denatured conditions as determined by high speed equilibrium centrifugation. An apparent molecular weight of 73,000 was found by sodium dodecyl sulfate gel electrophoresis. The protein has an apparent alpha helix content of 34% as determined from its circular dichroism spectrum. The extinction coefficient at 280 nm was found to be 62,200 M-1 cm-1. Lysine is the COOH-terminal residue and the sequence at the NH2 terminus is alanylarginine. No evidence of terminal heterogeneity was observed. The amino acid composition of EF-G revealed no unusual amino acids or prosthetic groups, but was notable in that the protein contains only 6 cysteine residues. The maintenance of at least one of these cysteines in the reduced form is essential for activity, since the protein is rapidly inactivated upon removal of protecting thiol. Under some conditions, activity can be partly restored by re-addition of a thiol. The per cent activity of the protein was examined by an active site titration, the formation of the EF-G-ribosome-GDP-fusidic acid complex. The formation of 1 mol of complex/mol of EF-G showed that the protein is 100% active.