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

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 U∧ACG and U∧ACU (∧ 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 (U∧ACG and U∧ACU). 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.

MazEF Homologs in Symbiobacterium thermophilum Exhibit Cross-Neutralization with Non-Cognate MazEFs.

Toxin-antitoxin systems are preserved by nearly every prokaryote. The type II toxin MazF acts as a sequence-specific endoribonuclease, cleaving ribonucleotides at specific sequences that vary from three to seven bases, as has been reported in different host organisms to date. The present study characterized the MazEF module (MazEF-sth) conserved in the Symbiobacterium thermophilum IAM14863 strain, a Gram-negative syntrophic bacterium that can be supported by co-culture with multiple bacteria, including Bacillus subtilis. Based on a method combining massive parallel sequencing and the fluorometric assay, MazF-sth was determined to cleave ribonucleotides at the UACAUA motif, which is markedly similar to the motifs recognized by MazF from B. subtilis (MazF-bs), and by several MazFs from Gram-positive bacteria. MazF-sth, with mutations at conserved amino acid residues Arg29 and Thr52, lost most ribonuclease activity, indicating that these residues that are crucial for MazF-bs also play significant roles in MazF-sth catalysis. Further, cross-neutralization between MazF-sth and the non-cognate MazE-bs was discovered, and herein, the neutralization mechanism is discussed based on a protein-structure simulation via AlphaFold2 and multiple sequence alignment. The conflict between the high homology shared by these MazF amino acid sequences and the few genetic correlations among their host organisms may provide evidence of horizontal gene transfer.