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Updated: May 20, 2026

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis
09:04

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

Published on: July 26, 2018

A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair

Birthe Meineke1, Alene Kast, Beate Schwer

  • 1Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.

RNA (New York, N.Y.)
|July 28, 2012
PubMed

Insights

Pichia acaciae toxin (PaT) and Kluyveromyces lactis zymocin are ribotoxins that deplete tRNAs in yeast. Their PaOrf2 and γ-toxin subunits use a two-incision mechanism to damage tRNA anticodons, evading repair.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Yeast Genetics

Background:

  • Pichia acaciae toxin (PaT) and Kluyveromyces lactis zymocin are secreted ribotoxins.
  • These toxins, specifically their PaOrf2 and γ-toxin subunits, function as tRNA anticodon nucleases.
  • They are assimilated by Saccharomyces cerevisiae, leading to growth arrest via specific tRNA depletion.

Purpose of the Study:

  • To investigate the molecular mechanism of PaOrf2 toxicity in Saccharomyces cerevisiae.
  • To identify key amino acid residues in PaOrf2 essential for its ribotoxicity and RNA transesterification activity.
  • To elucidate the tRNA cleavage and repair dynamics involving PaOrf2 and cellular/exogenous repair systems.

Main Methods:

  • Site-directed mutagenesis of PaOrf2 to identify critical residues (Glu9, His287).
  • Expression of PaOrf2 and γ-toxin in Saccharomyces cerevisiae to study toxicity.
  • Analysis of tRNA cleavage sites and the role of wobble nucleoside modifications (mcm(5)).
  • Complementation assays using phage and plant tRNA repair systems (tRNA ligases) in yeast mutants (elp3).

Main Results:

  • PaOrf2 residues Glu9 and His287 are crucial for toxicity, suggesting a conserved catalytic mechanism with γ-toxin.
  • PaOrf2 performs sequential incisions in tRNA: one 3' to the mcm(5)s(2)U wobble nucleoside, dependent on mcm(5), and a second upstream, excising a dinucleotide.
  • Phage and plant tRNA repair systems can partially rescue toxicity in elp3 cells (lacking mcm(5) modification) by repairing single-site cleavage.
  • Endogenous yeast tRNA ligase (Trl1) is insufficient for complete repair; plant tRNA ligase can provide complementary sealing activity.
  • The two-incision mechanism of PaOrf2 effectively evades productive repair by tRNA ligases, as complete damage prevents repair.

Conclusions:

  • PaOrf2 and γ-toxin share a similar RNA transesterification mechanism despite structural differences.
  • The two-incision cleavage strategy employed by PaOrf2 is a potent mechanism for tRNA anticodon damage.
  • This damage evades cellular tRNA repair pathways, highlighting a sophisticated evasion strategy by the toxin.

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