![]() Since then, the recognition sequences of various MazF homologs have been characterized ( Zhang et al., 2005 Zhu et al., 2009 Park et al., 2011 Rothenbacher et al., 2012 Schuster et al., 2013 Miyamoto et al., 2016a, 2017). Furthermore, binding of MazE -MazF complex or MazE to the MazEF promoter is lost, which relieves transcriptional repression ( Zhang et al., 2003a).Įscherichia coli MazF (MazF-Ec), consisting of 111 amino acids, was first reported to specifically cleave intracellular single-stranded mRNAs with ACA sequences ( Aizenman et al., 1996 Zhang et al., 2003b). Free MazF specifically cleaves target sequences within mRNAs, tRNAs, and rRNAs ( Zhang et al., 2003b Schifano et al., 2013, 2014, 2016). However, under stress conditions, MazE is degraded by the intracellular protease, thereby releasing MazF ( Aizenman et al., 1996 Muthuramalingam et al., 2016). Further, both the MazE -MazF complex and MazE can regulate MazEF transcription by binding to its TA promoter ( Zhang et al., 2003a). Under normal conditions, MazE binds to MazF to neutralize its ribonuclease activity ( Zhang et al., 2003a). The toxin MazF functions as a ribosome-independent single-stranded RNA endoribonuclease, while the cognate antitoxin MazE suppresses MazF activity ( Engelberg-Kulka et al., 2005). The type II TA system MazEF is one of the most extensively studied TA modules. In the type II TA systems, the toxin molecule is a stable protein and the antitoxin is a small unstable protein. TA systems are classified into six types (types I–VI) based on the nature and function of toxin and antitoxin molecules ( Unterholzner et al., 2013 Lee and Lee, 2016 Lobato-Márquez et al., 2016). Microorganisms harboring this system can control their growth and survival under various environmental conditions, such as starvation, oxidative stress, pathogen infection, and heat stress ( Fraikin et al., 2020). Highly conserved toxin-antitoxin (TA) systems are found on microbial plasmids and chromosomes and are specific to prokaryotes ( Yamaguchi et al., 2011). Our study posits UACAAA as the recognition sequence of MazF-Da and provides insight into the amino acid sites that are key to its unique enzymatic properties. This mutant cleaved UACAAG sequences in addition to UACAAA sequences, but did not cleave UACGAA or UACUAA sequences, suggesting that Asn36 affects the loosening and narrowing of MazF-Da cleavage sequence recognition. The recognition sequence of the N36A mutant differed from that of the wild type. Analysis using mutants with a single mutation at an amino acid residue site that is well conserved across various MazF toxins showed that G18, E20, R25, and P26 were important for the ribonuclease activity of MazF-Da. We found that MazF-Da exhibited the highest activity at around 60☌. MazF-Da, expressed using a cell-free protein synthesis system, is a six-base-recognition-specific ribonuclease that preferentially cleaves UACAAA sequences and weakly cleaves UACGAA and UACUAA sequences. Herein, we characterized MazF obtained from Candidatus Desulforudis audaxviator (MazF-Da) and identified the amino acid residues necessary for its catalytic function. In recent studies, crystallographic analysis of MazFs from several species have suggested the presence of amino acid sites important for MazF substrate RNA binding and for its catalytic activity. The toxin MazF, belonging to the type II TA system, functions as a sequence-specific ribonuclease that recognizes 3 to 7 bases. The toxin-antitoxin (TA) system, inherent to various prokaryotes, plays a critical role in survival and adaptation to diverse environmental stresses. Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.Hiroko Tamiya-Ishitsuka, Masako Tsuruga, Naohiro Noda * and Akiko Yokota * ![]()
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