Structural Basis of mRNA Recognition and Cleavage by Toxin MazF and Its Regulation by Antitoxin MazE in Bacillus subtilis

Abstract

SUMMARY MazF is an mRNA interferase, which, upon activation during stress conditions, cleaves mRNAs in a sequence-specific manner, resulting in cellular growth arrest. During normal growth conditions, the MazF toxin is inactivated through binding to its cognate antitoxin, MazE. How MazF specifically recognizes its mRNA target and carries out cleavage and how the formation of the MazE-MazF complex inactivates MazF remain unclear. We present crystal structures of MazF in complex with mRNA substrate and antitoxin MazE in Bacillus subtilis. The structure of MazF in complex with uncleavable UUdUACAUAA RNA substrate defines the molecular basis underlying the sequence-specific recognition of UACAU and the role of residues involved in the cleavage through site-specific mutational studies. The structure of the heterohexameric (MazF)2-(MazE)2-(MazF)2 complex in Bacillus subtilis, supplemented by mutational data, demonstrates that the positioning of the C-terminal helical segment of MazE within the RNA-binding channel of the MazF dimer prevents mRNA binding and cleavage by MazF.

Structure, Mechanism, and Specificity of a Eukaryal tRNA Restriction Enzyme Involved in Self-Nonself Discrimination

“…5E). Thus, the immunity mechanism of the fungal ribotoxin system entails direct binding of the immunity factor to the toxin and masking of its nuclease activity, presumably via steric occlusion of the ribotoxin's RNA binding site by the antitoxin, as has been seen with other ribotoxin•antitoxin pairs (Luna-Chavez et al, 2006; Yajima et al, 2006; Graille et al,2004; Simanshu et al, 2013). …”

Wake me when it’s over – Bacterial toxin–antitoxin proteins and induced dormancy

“…72 Crystal structures were determined of B. subtilis MazF, including complexes of MazF with MazE (Figure 2(a)) or mRNA containing the uncleavable dUACAU sequence (Figure 2(b)). 73 The MazF-mRNA complex showed dUACAU bound to MazF with the bases projecting towards the dimer interface of the toxin and the phosphate backbone moieties projecting away from the surface. Residues R25 and T48, which are highly conserved among MazF homologues, form hydrogen bonds with the oxygen atoms of the scissile phosphate between dU3 and A4 of the mRNA and either R25A or T48A mutations resulted in a loss of toxicity.…”

“…(a) MazF toxin homodimers from B. subtilis (red and gold) are inhibited when bound to the C-terminal region of MazE antitoxin proteins (blue and cyan) (PDB ID: 4ME7). 73 (b) A dimeric structure of MazF from B. subtilis (red and gold) is in complex with RNA (cyan sticks) (PDB ID: 4MDX). 73 (c) A homodimer of the DNA gyrase subunit A dimerization domain (green and light green) is bound to a CcdB homodimer (red and gold) from E. coli (PDB ID: 1X75).…”

“…73 (b) A dimeric structure of MazF from B. subtilis (red and gold) is in complex with RNA (cyan sticks) (PDB ID: 4MDX). 73 (c) A homodimer of the DNA gyrase subunit A dimerization domain (green and light green) is bound to a CcdB homodimer (red and gold) from E. coli (PDB ID: 1X75). 74 (d) A ParE toxin homodimer from Caulobacter vibrioides (red and gold) is inhibited by a ParD antitoxin homodimer (blue and cyan) (PDB ID: 3KXE).…”

Cloaked dagger: tRNA slicing by an unlikely culprit

“…Yet VapC toxins have an entirely different amino acid sequence with no similarity to MazF toxins, use a completely different mechanism for catalysis that involves a PIN domain comprising 4 highly conserved acidic residues coordinated with a metal ion, and the structures of VapC toxins do not share any obvious features with the structures of MazF. [31][32][33][34][35][36][37][38] This conundrum will only be solved once a high resolution crystal structure of MazF-mt9 bound its tRNA substrate is obtained and compared to other MazF toxins as well as tRNA-cleaving VapC toxins.…”