Characterization of YafO, an Escherichia coli Toxin

• Published in: The Journal of biological chemistry Vol. 284; no. 38; pp. 25522 - 25531
• Main Authors: Zhang, Yonglong, Yamaguchi, Yoshihiro, Inouye, Masayori
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.09.2009; American Society for Biochemistry and Molecular Biology
• Subjects: Bacterial Toxins - genetics; Bacterial Toxins - metabolism; Codon, Initiator - genetics; Codon, Initiator - metabolism; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Operon - physiology; Protein Biosynthesis - physiology; Protein Synthesis, Post-Translational Modification, and Degradation; Ribosome Subunits, Large, Bacterial - genetics; Ribosome Subunits, Large, Bacterial - metabolism; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Transfer, Met - genetics; RNA, Transfer, Met - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.036624

YafO is a toxin encoded by the yafN-yafO antitoxin-toxin operon in the Escherichia coli genome. Our results show that YafO inhibits protein synthesis but not DNA or RNA synthesis. The in vivo [35S]methionine incorporation was inhibited within 5 min after YafO induction. In in vivo primer extension experiments with two different mRNAs, the specific cleavage bands appeared 11–13 bases downstream of the initiation codon, AUG, 2.5 min after the induction of YafO. An identical band was also detected in in vitro toeprinting experiments when YafO was added to the reaction mixture containing 70 S ribosomes and the same mRNAs even in the absence of tRNAfMet. Notably, this band was not detected in the presence of YafO alone, indicating that YafO by itself does not have endoribonuclease activity under the conditions used. The full-length mRNAs almost completely disappeared 30 min after YafO induction in in vivo primer extension experiments, consistent with Northern blotting analysis. Over 84% of [35S]methionine-tRNAfMet was released from the translation initiation complex at 5.43 μm YafO in vitro. We demonstrated that the 70 S ribosome peak significantly increased upon YafO induction, and when the 70 S ribosomes dissociated into 50 and 30 S subunits, YafO was found to be associated with 50 S subunits. These results demonstrate that YafO is a ribosome-dependent mRNA interferase inhibiting protein synthesis.