Distinct paths to stop codon reassignment by the variant-code organisms Tetrahymena and Euplotes.

Area of Science:

• Molecular Biology
• Genetics
• Evolutionary Biology

Background:

• Stop codon reassignment, altering the genetic code, is prevalent in ciliate protozoa.
• Specific ciliate species like Tetrahymena and Euplotes exhibit unique stop codon recognition patterns.
• Domain 1 of the translation termination factor eRF1 is implicated in mediating stop codon recognition.

Purpose of the Study:

• To experimentally validate the role of eRF1 domain 1 in vivo for altered stop codon recognition in ciliates.
• To investigate whether domain 1 of ciliate eRF1s is the sole determinant of their unique stop codon usage.
• To compare the mechanisms by which Tetrahymena and Euplotes diverged from the universal genetic code.

Main Methods:

• Construction of hybrid eRF1 proteins by fusing domain 1 from Tetrahymena thermophila or Euplotes octocarinatus with domains 2 and 3 from Saccharomyces cerevisiae.
• Expression of these hybrid eRF1 proteins in yeast (Saccharomyces cerevisiae) cells.
• In vivo analysis of translation termination efficiency at different stop codons (UAA, UAG, UGA) using the hybrid proteins.

Main Results:

• The Tetrahymena hybrid eRF1 demonstrated efficient termination at all three stop codons in yeast, suggesting domain 1 is not the sole determinant.
• The Euplotes hybrid eRF1 facilitated efficient termination at UAA and UAG but not at UGA.
• These findings highlight that while eRF1 domain 1 is crucial, other factors modulate stop codon recognition.

Conclusions:

• Domain 1 of eRF1 is a key factor, but not the only one, in determining stop codon specificity in ciliates.
• Tetrahymena and Euplotes have evolved distinct molecular strategies to achieve their unique stop codon reassignments.
• This study provides in vivo evidence for the role of eRF1 domain 1 in genetic code evolution.