The histidine residue of codon 715 is essential for function of elongation factor 2.
Several mutant cDNAs of elongation factor 2 (EF-2) were constructed by site-directed mutagenesis and their products expressed in mouse cells were investigated. Amino acid substitution for the histidine residue of codon 715, which is modified post-translationally to diphthamide, resulted in non-functional EF-2 and this substitution did not render EF-2 resistant to Pseudomonas aeruginosa exotoxin A, which inactivates EF-2 transferring ADP-ribose to the diphthamide residue. These non-functional EF-2s with replacements of the histidine-715 residue showed various extents of inhibition of protein synthesis by competing with functional EF-2 in vivo. These results suggest that histidine-715 is essential for the translocase activity of EF-2 and that the region around diphthamide functions in recognition of, and/or binding to ribosomes. Substitution of proline for the alanine-713 residue and substitution of glutamine for the glycine-717 residue converted EF-2 to partially toxin-resistant forms. Two-dimensional gel analysis with fragment A of diphtheria toxin of these toxin-resistant EF-2s revealed that their ADP-ribosylations by toxin were much less than that of wild-type EF-2.
Omura
F
F
Institute for Molecular and Cellular Biology, Osaka University, Japan.
Kohno
K
K
Uchida
T
T
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D003062:Codon / Q000032:analysis*; D004247:DNA / Q000096:biosynthesis; D004351:Drug Resistance; D006639:Histidine / Q000031:analogs & derivatives / Q000032:analysis* / Q000378:metabolism; D007739:L Cells; D051379:Mice; D009154:Mutation; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000235:genetics / Q000502:physiology*; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis*; D014118:Toxins, Biological / Q000494:pharmacology
D003062:Codon; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D012333:RNA, Messenger; D014118:Toxins, Biological; D000246:Adenosine Diphosphate Ribose; D006639:Histidine; C027527:diphthamide; D004247:DNA