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. 1994 Apr 15;13(8):1960–1968. doi: 10.1002/j.1460-2075.1994.tb06465.x

Mechanism of post-segregational killing: Sok antisense RNA interacts with Hok mRNA via its 5'-end single-stranded leader and competes with the 3'-end of Hok mRNA for binding to the mok translational initiation region.

T Thisted 1, N S Sørensen 1, E G Wagner 1, K Gerdes 1
PMCID: PMC395038  PMID: 8168493

Abstract

The hok/sok system of plasmid R1, which mediates plasmid stabilization by killing of plasmid-free segregants, codes for two RNA species, Hok mRNA and Sok antisense RNA. The lethal expression of hok is inhibited post-transcriptionally by the 67 nt Sok-RNA. In this paper, we analyse the secondary structure of Sok-RNA and the binding of Sok-RNA to Hok mRNA in vitro. The reaction between the two RNAs leads to the formation of a complete duplex in which Sok-RNA is hybridized over its entire length to Hok mRNA. The second-order rate constant of duplex formation was determined to be approximately 1 x 10(5) M-1s-1. Mutations in the 5'-end single-stranded leader of Sok-RNA severely reduced the binding rate to wt Hok mRNA, whereas loop mutations in Sok-RNA had no such effect. The reduced binding rates were paralleled by abolished in vivo regulatory properties. These results suggest that, unlike in other well-characterized antisense/target RNA systems, the initial recognition reaction between Sok-RNA and Hok mRNA takes place between the single-stranded 5'-end of Sok-RNA and the complementary region in Hok mRNA, without the involvement of an antisense loop in the initial binding step. Furthermore, the finding that Sok-RNA competes with the 3'-end of full-length Hok mRNA for binding to the mok translational initiation region adds to the complexity of killer gene regulation.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Case C. C., Roels S. M., Jensen P. D., Lee J., Kleckner N., Simons R. W. The unusual stability of the IS10 anti-sense RNA is critical for its function and is determined by the structure of its stem-domain. EMBO J. 1989 Dec 20;8(13):4297–4305. doi: 10.1002/j.1460-2075.1989.tb08616.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Gerdes K., Larsen J. E., Molin S. Stable inheritance of plasmid R1 requires two different loci. J Bacteriol. 1985 Jan;161(1):292–298. doi: 10.1128/jb.161.1.292-298.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Gerdes K., Nielsen A., Thorsted P., Wagner E. G. 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. J Mol Biol. 1992 Aug 5;226(3):637–649. doi: 10.1016/0022-2836(92)90621-p. [DOI] [PubMed] [Google Scholar]
  4. Gerdes K., Poulsen L. K., Thisted T., Nielsen A. K., Martinussen J., Andreasen P. H. The hok killer gene family in gram-negative bacteria. New Biol. 1990 Nov;2(11):946–956. [PubMed] [Google Scholar]
  5. Gerdes K., Rasmussen P. B., Molin S. Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells. Proc Natl Acad Sci U S A. 1986 May;83(10):3116–3120. doi: 10.1073/pnas.83.10.3116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gerdes K., Thisted T., Martinussen J. Mechanism of post-segregational killing by the hok/sok system of plasmid R1: sok antisense RNA regulates formation of a hok mRNA species correlated with killing of plasmid-free cells. Mol Microbiol. 1990 Nov;4(11):1807–1818. doi: 10.1111/j.1365-2958.1990.tb02029.x. [DOI] [PubMed] [Google Scholar]
  7. Heus H. A., Pardi A. Structural features that give rise to the unusual stability of RNA hairpins containing GNRA loops. Science. 1991 Jul 12;253(5016):191–194. doi: 10.1126/science.1712983. [DOI] [PubMed] [Google Scholar]
  8. Hjalt T., Wagner E. G. The effect of loop size in antisense and target RNAs on the efficiency of antisense RNA control. Nucleic Acids Res. 1992 Dec 25;20(24):6723–6732. doi: 10.1093/nar/20.24.6723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kittle J. D., Simons R. W., Lee J., Kleckner N. Insertion sequence IS10 anti-sense pairing initiates by an interaction between the 5' end of the target RNA and a loop in the anti-sense RNA. J Mol Biol. 1989 Dec 5;210(3):561–572. doi: 10.1016/0022-2836(89)90132-0. [DOI] [PubMed] [Google Scholar]
  10. Nielsen A. K., Thorsted P., Thisted T., Wagner E. G., Gerdes K. The rifampicin-inducible genes srnB from F and pnd from R483 are regulated by antisense RNAs and mediate plasmid maintenance by killing of plasmid-free segregants. Mol Microbiol. 1991 Aug;5(8):1961–1973. doi: 10.1111/j.1365-2958.1991.tb00818.x. [DOI] [PubMed] [Google Scholar]
  11. Nordström K., Wagner E. G., Persson C., Blomberg P., Ohman M. Translational control by antisense RNA in control of plasmid replication. Gene. 1988 Dec 10;72(1-2):237–240. doi: 10.1016/0378-1119(88)90148-5. [DOI] [PubMed] [Google Scholar]
  12. Persson C., Wagner E. G., Nordström K. Control of replication of plasmid R1: formation of an initial transient complex is rate-limiting for antisense RNA--target RNA pairing. EMBO J. 1990 Nov;9(11):3777–3785. doi: 10.1002/j.1460-2075.1990.tb07591.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Persson C., Wagner E. G., Nordström K. Control of replication of plasmid R1: kinetics of in vitro interaction between the antisense RNA, CopA, and its target, CopT. EMBO J. 1988 Oct;7(10):3279–3288. doi: 10.1002/j.1460-2075.1988.tb03195.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Persson C., Wagner E. G., Nordström K. Control of replication of plasmid R1: structures and sequences of the antisense RNA, CopA, required for its binding to the target RNA, CopT. EMBO J. 1990 Nov;9(11):3767–3775. doi: 10.1002/j.1460-2075.1990.tb07590.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rasmussen P. B., Gerdes K., Molin S. Genetic analysis of the parB+ locus of plasmid R1. Mol Gen Genet. 1987 Aug;209(1):122–128. doi: 10.1007/BF00329846. [DOI] [PubMed] [Google Scholar]
  16. Simons R. W., Kleckner N. Biological regulation by antisense RNA in prokaryotes. Annu Rev Genet. 1988;22:567–600. doi: 10.1146/annurev.ge.22.120188.003031. [DOI] [PubMed] [Google Scholar]
  17. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Thisted T., Gerdes K. Mechanism of post-segregational killing by the hok/sok system of plasmid R1. Sok antisense RNA regulates hok gene expression indirectly through the overlapping mok gene. J Mol Biol. 1992 Jan 5;223(1):41–54. doi: 10.1016/0022-2836(92)90714-u. [DOI] [PubMed] [Google Scholar]
  19. Tomizawa J. Control of ColE1 plasmid replication: the process of binding of RNA I to the primer transcript. Cell. 1984 Oct;38(3):861–870. doi: 10.1016/0092-8674(84)90281-2. [DOI] [PubMed] [Google Scholar]
  20. Tomizawa J., Itoh T. Plasmid ColE1 incompatibility determined by interaction of RNA I with primer transcript. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6096–6100. doi: 10.1073/pnas.78.10.6096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Woese C. R., Winker S., Gutell R. R. Architecture of ribosomal RNA: constraints on the sequence of "tetra-loops". Proc Natl Acad Sci U S A. 1990 Nov;87(21):8467–8471. doi: 10.1073/pnas.87.21.8467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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