Insights into the specificity of RNA cleavage by the Escherichia coli MazF toxin

Abstract

The mazEF (chpA) toxin-antitoxin system of Escherichia coli is involved in the cell response to nutritional and antibiotic stresses as well as in bacterial-programmed cell death. Valuable information on the MazF toxin was derived from the determination of the crystal structure of the MazE/MazF complex and from in vivo data, suggesting that MazF promoted ribosome-dependent cleavage of messenger RNA. However, it was concluded from recent in vitro analyses using a MazF-(His6) fusion protein that MazF was an endoribonuclease that cleaved messenger RNA specifically at 5 0 -ACA-3 0 sites situated in singlestranded regions. In contrast, our work reported here shows that native MazF protein cleaves RNA at the 5 0 side of residue A in 5 0 -NAC-3 0 sequences (where N is preferentially U or A). MazFdependent cleavage occurred at target sequences situated either in single-or double-stranded RNA regions. These activities were neutralized by a His6-MazE antitoxin. Although essentially consistent with previous in vivo reports on the substrate specificity of MazF, our results strongly suggest that the endoribonuclease activity of MazF may be modulated by additional factors to cleave messenger and other cellular RNAs.

Toxin–antitoxin systems are ubiquitous and plasmid-encoded in vancomycin-resistant enterococci

“…MazE is a labile protein that is degraded by the ClpA protease; thus, in the absence of the genes encoding mazEF, the cellular MazE concentration drops rapidly, leaving MazF to kill or inhibit the growth of the cell (51). The MazF toxin is an endoribonuclease that is specific for ACA sequences (52)(53)(54), and its degradation of mRNA in this manner is believed to lead to the observed bacterial cell death (55). Although mazEF orthologs with varying degrees of homology are present on other bacterial chromosomes (31), including three mazEF homologs on the E. faecalis V583 genome and one on the V583 plasmid pTEF1 (33), the PCR products investigated in this study show surprisingly strong sequence similarity to the mazEF genes from the E. coli K12 genome.…”

Functional Identification of Toxin-Antitoxin Molecules from Helicobacter pylori 26695 and Structural Elucidation of the Molecular Interactions

“…These toxins function, or are supposed to function, by inhibiting translation through mRNA cleavage (6). Among those toxins, YoeB, YafQ, MazF, and ChpBK have intrinsic RNase activity (12)(13)(14)(15)(16), whereas RelE does not. RelE is a ribosome-dependent endoribonuclease that is active when it associates with a ribosome (17,18) or a stimulatory factor for the intrinsic endonuclease activity of a ribosome (12,19).…”

Structure and Function of Bacterial Kid-Kis and Related Toxin-Antitoxin Systems

“…Their result closely resembles the RNA-binding site of Kid in our Kid-RNA complex [60]. The antitoxin MazE prevents RNA cleavage by MazF [77,79] by binding to the substratebinding site of the toxin [43,80].…”

“…Like Kid, MazF has been shown to inhibit protein synthesis in cell extracts and act as an endoribonuclease able to cleave RNA in the absence of ribosomes in vitro [77][78][79]. This toxin was reported either to cleave XACA sequences at the 5' end of ACA yielding a 2':3'-cyclic phosphate at one side and a free 5'-OH group at the other side [77,78], or to cleave at the 5' end of residue A in NAC sites (where N is U or A) [79]. The 2'-hydroxyl group of de nucleotide preceding the adenosine was shown to be essential for RNA cleavage [78].…”

“…As for Kid, the precise nature of the MazF substrate is subject to discussion. It could be only single-stranded mRNAs [77,78], or alternatively, all cellular RNAs including both single-and doublestranded RNA [79]. Ikura and co-workers used a singlestranded DNA fragment in a NMR titration experiment to map the substrate-binding face of MazF [80].…”