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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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One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
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Short tRNA anticodon stem and mutant eRF1 allow stop codon reassignment.

Ambar Kachale1,2, Zuzana Pavlíková3, Anna Nenarokova1,2,4

  • 1Institute of Parasitology, Biology Centre, Czech Academy of Sciences, České Budějovice, Czech Republic.

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|January 11, 2023
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Summary
This summary is machine-generated.

Some protists reassign stop codons. This study reveals a universal mechanism involving tRNA modifications and release factor mutations in eukaryotes, enabling stop codon reassignment and gene expression regulation.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Evolutionary Biology

Background:

  • The standard genetic code uses specific codons as stop signals during translation.
  • Some organisms, particularly protists, have reassigned these stop codons to encode amino acids, altering fundamental biological principles.
  • Understanding the mechanisms behind stop codon reassignment is crucial for comprehending gene expression and evolution.

Purpose of the Study:

  • To investigate the mechanisms of stop codon reassignment in the previously undescribed trypanosomatid, Blastocrithidia nonstop.
  • To identify the specific genetic and molecular adaptations that allow for the use of stop codons as sense codons.
  • To explore the evolutionary conservation and universality of these mechanisms in eukaryotes.

Main Methods:

  • Analysis of in-frame stop codons in 7,259 protein-coding genes of Blastocrithidia nonstop.
  • Identification and characterization of novel tRNAs (tRNAGlu, tRNATrp) involved in stop codon reassignment.
  • Engineering and expression of tRNA variants in different species (B. nonstop, Trypanosoma brucei, Saccharomyces cerevisiae) to study their function.
  • Mutation analysis of release factor 1 in B. nonstop.

Main Results:

  • In-frame stop codons are underrepresented in highly expressed genes in B. nonstop; UAA is the sole termination codon.
  • Reassignment of UAG and UAA codons involved new cognate tRNAsGlu.
  • UGA reassignment occurred via a shortened (4 base pairs) anticodon stem of tRNATrpCCA, enabling tryptophan incorporation.
  • Engineered 4-bp tRNATrp variants showed increased readthrough in multiple species.
  • A mutated release factor 1 in B. nonstop specifically restricted UGA recognition, enhancing reassignment.

Conclusions:

  • Blastocrithidia nonstop utilizes a unique mechanism for UGA stop codon reassignment through tRNA anticodon stem shortening.
  • A modified release factor 1 further potentiates UGA reassignment in B. nonstop.
  • Similar strategies involving tRNA modifications and release factor alterations are employed by other eukaryotes like Condylostoma magnum.
  • A previously unknown, universal mechanism for stop codon reassignment has been identified in unrelated eukaryotes.