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Base complementarity between the three base pairs of mRNA codon and the tRNA anticodon is not a failsafe mechanism. Inaccuracies can range from a single mismatch to no correct base pairing at all. The free energy difference between the correct and nearly correct base pairs can be as small as 3 kcal/ mol. With complementarity being the only proofreading step, the estimated error frequency would be one wrong amino acid in every 100 amino acids incorporated. However, error frequencies observed in...
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Point mutations are genetic alterations involving the change of a single nucleotide base pair in DNA. Depending on how the alteration affects protein synthesis, they can lead to various consequences.Point mutations fall into the following types:Silent mutations occur when a nucleotide change does not alter the amino acid sequence due to the redundancy of the genetic code. For instance, changing ACC to ACA still encodes threonine, leaving the protein function unaffected. This occurs because...
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Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
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During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R...
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eEF2 diphthamide modification restrains spurious frameshifting to maintain translational fidelity.

Byung-Sik Shin1, Ivaylo P Ivanov1, Joo-Ran Kim1

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The diphthamide modification on translation factor eEF2 is crucial for maintaining translational fidelity, preventing ribosomal frameshifting and premature termination. Its absence impairs protein synthesis accuracy, despite not being essential for cell survival.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Diphthamide (DPH) is a unique post-translational modification of elongation factor 2 (eEF2).
  • DPH is essential for resistance to bacterial toxins but its precise cellular function remains unclear.
  • DPH is synthesized through a complex enzymatic pathway in eukaryotes.

Purpose of the Study:

  • To investigate the cellular function of diphthamide (DPH) modification.
  • To characterize the impact of DPH deficiency on translation fidelity and cellular processes.
  • To understand the evolutionary significance of DPH.

Main Methods:

  • Analysis of Saccharomyces cerevisiae mutants lacking DPH.
  • Ribosome profiling in yeast and mammalian cells.
  • Assessing ribosomal frameshifting and mRNA translation elongation.
  • Investigating the effect of DPH ADP-ribosylation on eEF2-ribosome interaction.

Main Results:

  • Loss of DPH increases resistance to sordarin and enhances -1 ribosomal frameshifting.
  • DPH deficiency leads to increased ribosomal drop-off and premature termination.
  • ADP-ribosylation of DPH hinders eEF2 binding to ribosomes, impairing translation.
  • Removal of out-of-frame stop codons rescues ribosomal processivity on long mRNAs.

Conclusions:

  • Diphthamide modification is vital for maintaining translational fidelity during elongation.
  • Loss of DPH results in widespread ribosomal frameshifting and premature termination.
  • DPH likely evolved to ensure accurate protein synthesis, despite its vulnerability to toxins.