Studies on the Interaction between Ribosome and Elongation Factor 2 by Fluorescent Labeling of the Diphtamide Residue

Abstract

The elongation factor 2 (EF-2) catalyzes translocation, which is the last step of the elongation cycle in the course of protein synthesis. The factor is the only known protein that is ADP-ribosylated by diphtheria toxin in the presence of NAD. The target of this reaction is a post-translationally modified histidine residue, called diphthamide. ADP-ribosylated EF-2 is unable to translocate, but still retains the ability to form a ternary complex with GDP and ribosome (1). We demonstrated that diphtheria toxin is able to recognize as a substrate an NAD analog, nicotinamide 1,N⁶ ethenoadeninedinucleotide (ε NAD), and to transfer the fluorescent ε ADP-ribose moiety onto the EF-2 diphthamide residue (2). Therefore, it was possible to label the EF-2 with this fluorescent probe by an efficient and selective enzymatic mechanism which allowed us to confidently attribute the molecular localization of the fluorophore at the level of the diphthamide residue.