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Substrate specificity of Mycobacterium tuberculosis tRNA terminal nucleotidyltransferase toxin MenT3

Jun Liu1, Yuka Yashiro1, Yuriko Sakaguchi2

  • 1Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan.

Nucleic Acids Research
|March 14, 2024
PubMed

Insights

Mycobacterium tuberculosis MenT3 toxin adds nucleotides to tRNA, inhibiting protein synthesis. It specifically targets seryl-tRNA, with its N-terminal region and tRNA

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Structural Biology

Background:

  • Mycobacterium tuberculosis transfer RNA (tRNA) terminal nucleotidyltransferase toxin (MenT3) inhibits protein synthesis by modifying tRNA.
  • MenT3's precise mechanism of action and substrate specificity were not fully understood.

Purpose of the Study:

  • To elucidate the nucleotide specificity and structural basis of MenT3 activity.
  • To identify the specific tRNA targets of MenT3 and the molecular determinants of this specificity.

Main Methods:

  • X-ray crystallography to determine the MenT3-CTP complex structure.
  • Biochemical assays to assess nucleotide incorporation into various tRNA species.
  • Site-directed mutagenesis and chimeric tRNA construction to investigate structure-function relationships.

Main Results:

  • MenT3 preferentially incorporates cytidine monophosphate (CMP) into the 3'-CCA end of tRNA, with a CTP-specific binding pocket identified in its crystal structure.
  • MenT3 selectively reduces seryl-tRNA levels in E. coli, efficiently incorporating CMPs into tRNASer.
  • The longer variable loop of class II tRNASer and MenT3's N-terminal region are crucial for substrate recognition and efficient CMP incorporation.

Conclusions:

  • MenT3 acts as a CTP-specific tRNA nucleotidyltransferase that inhibits protein synthesis by targeting specific tRNAs, particularly tRNASer.
  • The structural and functional data reveal a mechanism for MenT3's tRNA substrate selection involving interactions between its N-terminal region and the tRNA's variable loop.
  • Understanding MenT3's mechanism provides insights into tRNA modification and its role in bacterial pathogenesis.

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