Photo-cross-linking of guanine nucleotides to the nucleotide binding site of elongation factor G

Abstract

Elongation factor G (EF-G) is rapidly inactivated when irradiated at 253.7 nm. The inactivation follows first-order single-hit kinetics with a quantum efficiency of 3.15 × 10 −5 μmol/μE. Inclusion of either GTP or GDP in the irradiation mixture does not alter the kinetics of inactivation, but does result in the covalent attachment of nucleotide to between 10 and 20% of the EF-G. This relatively low percentage of cross-linking is due to the rapid rate of photoinactivation as compared to the slower rate of covalent attachment. If EF-G is reacted before irradiation with N-ethylmaleimide, a modification known to block the nucleotide binding site [ Rohrbach and Bodley (1976) J. Biol. Chem. 251, 930 ], essentially no nucleotide can be photo-cross-linked to EF-G. Treatment of the photo-cross-linked GTP-EF-G with Raney nickel led to the liberation of the nucleotide moiety, indicating that the photo-cross-link to EF-G occurred through a sulfur atom. Although the formation of the EF-G nucleotide complex has been shown to be an obligatory first step in the formation of the EF-G nucleotide ribosome complex [ Rohrbach and Bodley (1976) Biochemistry 15, 4565 ], the covalent EF-G-nucleotide adduct cannot form a ternary complex with the ribosome. The presence of both nucleotide and ribosomes during irradiation drastically alters the kinetics of inactivation. The inactivation under these conditions follows multiple-hit kinetics with an initial period during which no EF-G activity is lost. Following this lag period, EF-G is inactivated at the same rate at which ribosomes lose their ability to bind EF-G. No nucleotide is cross-linked to EF-G or the ribosome under these conditions.