title: The structure and function ofMycobacterium tuberculosisMazF-mt6 toxin provide insights into conserved features of MazF endonucleases.
abstract: Toxin-antitoxin systems are ubiquitous in prokaryotic and archaeal genomes and regulate growth in response to stress.Escherichia colicontains at least 36 putative toxin-antitoxin gene pairs, and some pathogens such asMycobacterium tuberculosishave over 90 toxin-antitoxin operons.E. coliMazF cleaves free mRNA after encountering stress, and nineM. tuberculosisMazF family members cleave mRNA, tRNA, or rRNA. Moreover,M. tuberculosisMazF-mt6 cleaves 23S rRNA Helix 70 to inhibit protein synthesis. The overall tertiary folds of these MazFs are predicted to be similar, and therefore, it is unclear how they recognize structurally distinct RNAs. Here we report the 2.7-Å X-ray crystal structure of MazF-mt6. MazF-mt6 adopts a PemK-like fold but lacks an elongated β1-β2 linker, a region that typically acts as a gate to direct RNA or antitoxin binding. In the absence of an elongated β1-β2 linker, MazF-mt6 is unable to transition between open and closed states, suggesting that the regulation of RNA or antitoxin selection may be distinct from other canonical MazFs. Additionally, a shortened β1-β2 linker allows for the formation of a deep, solvent-accessible, active-site pocket, which may allow recognition of specific, structured RNAs like Helix 70. Structure-based mutagenesis and bacterial growth assays demonstrate that MazF-mt6 residues Asp-10, Arg-13, and Thr-36 are critical for RNase activity and likely catalyze the proton-relay mechanism for RNA cleavage. These results provide further critical insights into how MazF secondary structural elements adapt to recognize diverse RNA substrates.
authors.affiliation: From the Biochemistry, Cell and Developmental Biology Program, Graduate Division of Biological and Biomedical Sciences and.|the Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
authors.forename: Eric D
authors.initials: ED
authors.lastname: Hoffer
authors.affiliation: the Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
authors.forename: Stacey J
authors.initials: SJ
authors.lastname: Miles
authors.affiliation: the Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322 christine.m.dunham@emory.edu.
authors.forename: Christine M
authors.identifier: 0000-0002-8821-688X
authors.initials: CM
authors.lastname: Dunham
mesh_terms: D000992:Antitoxins / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009690:Nucleic Acid Conformation; D009876:Operon; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D000992:Antitoxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C422955:MazF protein, E coli; D012333:RNA, Messenger; D012338:RNA, Ribosomal, 23S; D004722:Endoribonucleases; C000601629:MazF protein, Mycobacterium tuberculosis
keywords: X-ray crystallography; bacterial toxin; endoribonuclease; protein synthesis; ribosomal ribonucleic acid (rRNA) (ribosomal RNA); ribosome