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. 1994 Aug 1;13(15):3599–3607. doi: 10.1002/j.1460-2075.1994.tb06667.x

Antisense RNA-mediated transcriptional attenuation occurs faster than stable antisense/target RNA pairing: an in vitro study of plasmid pIP501.

S Brantl 1, E G Wagner 1
PMCID: PMC395265  PMID: 7520390

Abstract

Antisense RNA-mediated transcriptional attenuation is the mode of replication control of several plasmids, among them pIP501. This mechanism implies that the repR mRNAs can fold into two mutually exclusive structures. The formation of one of these structures is induced by binding of the antisense RNA and results in premature termination. Since the fate of the nascent mRNA transcripts depends on the binding rate of the antisense RNA to its target, the control is kinetic. We have studied the antisense RNA, RNAIII, and target RNA, RNAII, whose interaction determines the replication frequency of plasmid pIP501. RNA secondary structures were analyzed using structure-specific RNases. RNA binding was studied in vitro with normal size and truncated RNAIII species. An in vitro single-round attenuation assay was developed that permits qualitative and quantitative assessment of inhibition by RNAIII. The effect of varying concentrations of RNAIII species on attenuation was tested and inhibition rate constants were calculated. The inhibition rate constants were at least 10 times higher than the pairing rate constants. Thus, steps preceding stable RNA duplex formation are sufficient to induce RNAIII-dependent termination of nascent RNAII transcripts.

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Selected References

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

  1. Asano K., Moriwaki H., Mizobuchi K. An induced mRNA secondary structure enhances repZ translation in plasmid ColIb-P9. J Biol Chem. 1991 Dec 25;266(36):24549–24556. [PubMed] [Google Scholar]
  2. Behnke D., Ferretti J. J. Physical mapping of plasmid pDB101: a potential vector plasmid for molecular cloning in streptococci. Plasmid. 1980 Sep;4(2):130–138. doi: 10.1016/0147-619x(80)90002-5. [DOI] [PubMed] [Google Scholar]
  3. Blomberg P., Nordström K., Wagner E. G. Replication control of plasmid R1: RepA synthesis is regulated by CopA RNA through inhibition of leader peptide translation. EMBO J. 1992 Jul;11(7):2675–2683. doi: 10.1002/j.1460-2075.1992.tb05333.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brantl S., Behnke D., Alonso J. C. Molecular analysis of the replication region of the conjugative Streptococcus agalactiae plasmid pIP501 in Bacillus subtilis. Comparison with plasmids pAM beta 1 and pSM19035. Nucleic Acids Res. 1990 Aug 25;18(16):4783–4790. doi: 10.1093/nar/18.16.4783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brantl S., Behnke D. Copy number control of the streptococcal plasmid pIP501 occurs at three levels. Nucleic Acids Res. 1992 Feb 11;20(3):395–400. doi: 10.1093/nar/20.3.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brantl S., Birch-Hirschfeld E., Behnke D. RepR protein expression on plasmid pIP501 is controlled by an antisense RNA-mediated transcription attenuation mechanism. J Bacteriol. 1993 Jul;175(13):4052–4061. doi: 10.1128/jb.175.13.4052-4061.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brenner M., Tomizawa J. Quantitation of ColE1-encoded replication elements. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):405–409. doi: 10.1073/pnas.88.2.405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carleton S., Projan S. J., Highlander S. K., Moghazeh S. M., Novick R. P. Control of pT181 replication II. Mutational analysis. EMBO J. 1984 Oct;3(10):2407–2414. doi: 10.1002/j.1460-2075.1984.tb02147.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clewell D. B., Yagi Y., Dunny G. M., Schultz S. K. Characterization of three plasmid deoxyribonucleic acid molecules in a strain of Streptococcus faecalis: identification of a plasmid determining erythromycin resistance. J Bacteriol. 1974 Jan;117(1):283–289. doi: 10.1128/jb.117.1.283-289.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eguchi Y., Tomizawa J. Complexes formed by complementary RNA stem-loops. Their formations, structures and interaction with ColE1 Rom protein. J Mol Biol. 1991 Aug 20;220(4):831–842. doi: 10.1016/0022-2836(91)90356-b. [DOI] [PubMed] [Google Scholar]
  11. Gerhart E., Wagner H., Nordström K. Structural analysis of an RNA molecule involved in replication control of plasmid R1. Nucleic Acids Res. 1986 Mar 25;14(6):2523–2538. doi: 10.1093/nar/14.6.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Horodniceanu T., Bouanchaud D. H., Bieth G., Chabbert Y. A. R plasmids in Streptococcus agalactiae (group B). Antimicrob Agents Chemother. 1976 Nov;10(5):795–801. doi: 10.1128/aac.10.5.795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Nordström K., Molin S., Light J. Control of replication of bacterial plasmids: genetics, molecular biology, and physiology of the plasmid R1 system. Plasmid. 1984 Sep;12(2):71–90. doi: 10.1016/0147-619x(84)90054-4. [DOI] [PubMed] [Google Scholar]
  16. Novick R. P., Iordanescu S., Projan S. J., Kornblum J., Edelman I. pT181 plasmid replication is regulated by a countertranscript-driven transcriptional attenuator. Cell. 1989 Oct 20;59(2):395–404. doi: 10.1016/0092-8674(89)90300-0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. Sørensen M. A., Jensen K. F., Pedersen S. High concentrations of ppGpp decrease the RNA chain growth rate. Implications for protein synthesis and translational fidelity during amino acid starvation in Escherichia coli. J Mol Biol. 1994 Feb 18;236(2):441–454. doi: 10.1006/jmbi.1994.1156. [DOI] [PubMed] [Google Scholar]
  22. Tamm J., Polisky B. Structural analysis of RNA molecules involved in plasmid copy number control. Nucleic Acids Res. 1983 Sep 24;11(18):6381–6397. doi: 10.1093/nar/11.18.6381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Thisted T., Sørensen N. S., Wagner E. G., Gerdes K. 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. EMBO J. 1994 Apr 15;13(8):1960–1968. doi: 10.1002/j.1460-2075.1994.tb06465.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tomizawa J. Control of ColE1 plasmid replication. Interaction of Rom protein with an unstable complex formed by RNA I and RNA II. J Mol Biol. 1990 Apr 20;212(4):695–708. doi: 10.1016/0022-2836(90)90231-a. [DOI] [PubMed] [Google Scholar]
  25. Tomizawa J. Control of ColE1 plasmid replication: binding of RNA I to RNA II and inhibition of primer formation. Cell. 1986 Oct 10;47(1):89–97. doi: 10.1016/0092-8674(86)90369-7. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Uhlin B. E., Nordström K. A runaway-replication mutant of plasmid R1drd-19: temperature-dependent loss of copy number control. Mol Gen Genet. 1978 Oct 4;165(2):167–179. doi: 10.1007/BF00269904. [DOI] [PubMed] [Google Scholar]
  28. Wilson I. W., Praszkier J., Pittard A. J. Mutations affecting pseudoknot control of the replication of B group plasmids. J Bacteriol. 1993 Oct;175(20):6476–6483. doi: 10.1128/jb.175.20.6476-6483.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wu R., Wang X., Womble D. D., Rownd R. H. Expression of the repA1 gene of IncFII plasmid NR1 is translationally coupled to expression of an overlapping leader peptide. J Bacteriol. 1992 Dec;174(23):7620–7628. doi: 10.1128/jb.174.23.7620-7628.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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