Two GTPs Are Required for Each Step in Elongation

The translocation reaction in E. coli. The translocation reaction occurs immediately after peptide synthesis. It involves displacement of the discharged tRNA from the P site and concerted movement of the peptidyl-tRNA and mRNA so that the peptidyl-tRNA is bound to the P site and the same three nucleotides in the mRNA. The A site is vacated and ready for the addition of another aminoacyl-tRNA. Translocation in eukaryotes is similar except that the EF-2 factor is involved instead of the EF-G factor. 

In the cytoplasm of the cell, the catalytic portion of diphtheria toxin acts as a very specific protein-modifying enzyme. It catalyzes the ADP-ribosylation and consequent inactivation of EF-2 by the following reaction  

This reaction is reversible when conducted in vitro, but under the conditions of pH and nicotinamide concentration that exist in the cell, it is irreversible. Thus, diphtheria toxin kills cells by irreversibly destroying the ability of EF-2 to participate in the translocation step of protein synthesis elongation. A number of other protein toxins have subsequently been found to ADP-ribosylate and inactivate cellular proteins involved in other essential cellular pathways. For example, cholera and pertussis toxins ADP-ribosylate and inactivate proteins important to cAMP metabolism. 

The enzymatic specificity of diphtheria toxin deserves special comment. The toxin ADP-ribosylates EF-2 in all eukaryotic cells in vitro whether or not they are sensitive to the toxin in vivo, but it does not modify any other protein, including the bacterial counterpart of EF-2. This narrow enzymatic specificity has called attention to an unusual posttranslational derivative of histidine, diphthamide, that occurs in EF-2 at the site of ADP-ribosylation (see fig. 1). Although the unique occurrence of diphthamide in EF-2 explains the specificity of the toxin, it raises questions about the functional significance of this modification in translocation. Interestingly, some mutants of eukaryotic cells selected for toxin resistance lack one of several enzymes necessary for the posttranslational synthesis of diphthamide in EF-2 that is necessary for toxin recognition, but these cells seem perfectly competent in protein synthesis. Thus, the raison d etre of diphthamide, as well as the biological origin of the toxin that modifies it, remains a mystery. 

Inactivation of the EF-2 factor by diphtheria toxin through the ADP-ribosylation of a modified histidine side chain. 

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Three Elongation Reactions Are Repeated with the Incorporation of Each Amino Acid Two GTPs Are Required for Each Step in Elongation... 

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