Translation elongation factor 2 anticodon mimicry domain mutants affect fidelity and diphtheria toxin resistance.
Eukaryotic elongation factor 2 (eEF2) mediates translocation in protein synthesis. The molecular mimicry model proposes that the tip of domain IV mimics the anticodon loop of tRNA. His-699 in this region is post-translationally modified to diphthamide, the target for Corynebacterium diphtheriae and Pseudomonas aeruginosa toxins. ADP-ribosylation by these toxins inhibits eEF2 function causing cell death. Mutagenesis of the tip of domain IV was used to assess both functions. A H694A mutant strain was non-functional, whereas D696A, I698A, and H699N strains conferred conditional growth defects, sensitivity to translation inhibitors, and decreased total translation in vivo. These mutant strains and those lacking diphthamide modification enzymes showed increased -1 frameshifting. The effects are not due to reduced protein levels, ribosome binding, or GTP hydrolysis. Functional eEF2 forms substituted in domain IV confer dominant diphtheria toxin resistance, which correlates with an in vivo effect on translation-linked phenotypes. These results provide a new mechanism in which the translational machinery maintains the accurate production of proteins, establishes a role for the diphthamide modification, and provides evidence of the ability to suppress the lethal effect of a toxin targeted to eEF2.
Ortiz
Pedro A
PA
Department of Molecular Genetics, Microbiology and Immunology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854-5635, USA.
Ulloque
Rory
R
Kihara
George K
GK
Zheng
Haiyan
H
Kinzy
Terri Goss
TG
D000926:Anticodon; D004167:Diphtheria Toxin / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D008958:Models, Molecular; D018716:Molecular Mimicry; D016296:Mutagenesis; D009154:Mutation; D010441:Peptide Chain Elongation, Translational; D020652:Peptide Elongation Factor 2 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D020539:Sequence Analysis, Protein
D000926:Anticodon; D004167:Diphtheria Toxin; D020652:Peptide Elongation Factor 2; D012343:RNA, Transfer