Arg-73 of the RNA endonuclease MazF in Salmonella enterica subsp. arizonae contributes to guanine and uracil recognition in the cleavage sequence

Takuma Okabe, Rie Aoi, Akiko Yokota, Hiroko Tamiya-Ishitsuka, Yunong Jiang, Akira Sasaki, Satoshi Tsuneda*, Naohiro Noda

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

The sequence-specific endoribonuclease MazF is widely conserved among prokaryotes. Approximately 20 different MazF cleavage sequences have been discovered, varying from three to seven nucleotides in length. Although MazFs from various prokaryotes were found, the cleavage sequences of most MazFs are unknown. Here, we characterized the conserved MazF of Salmonella enterica subsp. arizonae (MazF-SEA). Using massive parallel sequencing and fluorometric assays, we revealed that MazF-SEA preferentially cleaves the sequences UACG and UACU ( represents cleavage sites). In addition, we predicted the 3D structure of MazF-SEA using AlphaFold2 and aligned it with the crystal structure of RNA-bound Bacillus subtilis MazF to evaluate RNA interactions. We found Arg-73 of MazF-SEA interacts with RNAs containing G and U at the third position from the cleavage sites (UACG and UACU). We then obtained the mutated MazF-SEA R73L protein to evaluate the significance of Arg-73 interaction with RNAs containing G and U at this position. We also used fluorometric and kinetic assays and showed the enzymatic activity of MazF-SEA R73L for the sequence UACG and UACU was significantly decreased. These results suggest Arg-73 is essential for recognizing G and U at the third position from the cleavage sites. This is the first study to our knowledge to identify a single residue responsible for RNA recognition by MazF. Owing to its high specificity and ribosome-independence, MazF is useful for RNA cleavage in vitro. These results will likely contribute to increasing the diversity of MazF specificity and to furthering the application of MazF in RNA engineering.

Original languageEnglish
Article number105636
JournalJournal of Biological Chemistry
Volume300
Issue number2
DOIs
Publication statusPublished - 2024 Feb

Keywords

  • RNA-protein interaction
  • Salmonella enterica
  • endoribonuclease
  • enzyme kinetics
  • fluorescence resonance energy transfer (FRET)
  • protein engineering
  • structural model
  • substrate specificity

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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