Insights into the specificity of RNA cleavage by the Escherichia coli MazF toxin.
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'-ACA-3' sites situated in single-stranded regions. In contrast, our work reported here shows that native MazF protein cleaves RNA at the 5' side of residue A in 5'-NAC-3' sequences (where N is preferentially U or A). MazF-dependent 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.
Muñoz-Gómez
Ana J
AJ
Department of Molecular Microbiology, Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
Santos-Sierra
Sandra
S
Berzal-Herranz
Alfredo
A
Lemonnier
Marc
M
Díaz-Orejas
Ramón
R
D000818:Animals; D000992:Antitoxins / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004722:Endoribonucleases / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020836:Protein Structure, Quaternary; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013379:Substrate Specificity; D014461:Ultracentrifugation
D000992:Antitoxins; D001427:Bacterial Toxins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C422954:MazE protein, E coli; C422955:MazF protein, E coli; D012329:RNA, Bacterial; D004722:Endoribonucleases