title: Mechanism of killer gene activation. Antisense RNA-dependent RNase III cleavage ensures rapid turn-over of the stable hok, srnB and pndA effector messenger RNAs.
abstract: The hok/sok, srnB and pnd systems of plasmids R1, F and R438 mediate plasmid maintenance by killing plasmid-free segregants. The systems encode exceptionally stable full-length mRNAs that code for potent cell toxins that kill the cells from within. The systems also produce truncated mRNAs whose appearance is correlated with killing activity. The truncated mRNAs are shortened by 35 to 70 nucleotides in the 3' ends, but have the same 5' ends as the full-length transcripts. Translation of the stable killer mRNAs is regulated by unstable antisense RNAs that are complementary to the leader regions of the full-length and truncated mRNAs. We show here, that both the presence of the antisense RNA and of the host enzyme RNase III is required for rapid cleavage of the truncated mRNAs, and we map the cleavage point in the Hok mRNA in vitro and in vivo to be located between nucleotides +245 and +246. The RNase III cleavage products of the Hok mRNA were found to be very unstable in vivo. Thus, RNase III cleavage seems to be the initial event leading to decay of the killer mRNAs. In an rnc- strain, the truncated mRNA species were found in steady-state cells. This observation indicates that the truncated mRNAs are formed constitutively and independently of the presence of the antisense RNAs. Thus, the antisense RNAs prevent the accumulation of the truncated mRNAs solely by mediating their rapid hydrolysis by RNase III. Furthermore, the generation of the truncated killer mRNAs in the rnc- host indicate that RNase III is dispensable for induction of the killer gene systems. Based on these and on observations obtained previously, we present a molecular model that explains the activation of the killer mRNAs in plasmid-free segregants and after addition of rifampicin.
authors.affiliation: Department of Molecular Biology, Odense University, Denmark.
authors.forename: K
authors.initials: K
authors.lastname: Gerdes
authors.forename: A
authors.initials: A
authors.lastname: Nielsen
authors.forename: P
authors.initials: P
authors.lastname: Thorsted
authors.forename: E G
authors.initials: EG
authors.lastname: Wagner
mesh_terms: D001483:Base Sequence; D015152:Blotting, Northern; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005804:Genes, Lethal; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids; D014176:Protein Biosynthesis; D016372:RNA, Antisense / Q000235:genetics / Q000378:metabolism*; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043244:Ribonuclease III; D015533:Transcriptional Activation
chemical_list: D029968:Escherichia coli Proteins; D016372:RNA, Antisense; D012329:RNA, Bacterial; D012333:RNA, Messenger; D004722:Endoribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli