Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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The Terminal and Internal Hairpin Loops of the ctRNA of Plasmid pJB01 Play Critical Roles in Regulating Copy Number

  • Kim, Sam Woong (Division of Biological Sciences, Pusan National University) ;
  • Jeong, In Sil (Division of Applied Life Sciences (Brain Korea 21 and EBNCRC), Graduate School of Gyeongsang National University) ;
  • Jeong, Eun Ju (Division of Applied Life Sciences (Brain Korea 21 and EBNCRC), Graduate School of Gyeongsang National University) ;
  • Tak, Je Il (Division of Applied Life Sciences (Brain Korea 21 and EBNCRC), Graduate School of Gyeongsang National University) ;
  • Lee, John Hwa (College of Veterinary Medicine, Chonbuk National University) ;
  • Eo, Seong Kug (College of Veterinary Medicine, Chonbuk National University) ;
  • Kang, Ho Young (Division of Biological Sciences, Pusan National University) ;
  • Bahk, Jeong Dong (Division of Applied Life Sciences (Brain Korea 21 and EBNCRC), Graduate School of Gyeongsang National University)
  • Received : 2007.10.25
  • Accepted : 2008.01.14
  • Published : 2008.07.31
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Abstract

The plasmid pJB01, a member of the pMV158 family isolated from Enterococcus faecium JC1, contains three open reading frames, copA, repB, and repC. Plasmids included in this family produce counter-transcribed RNA (ctRNA) that contributes to copy number control. The pJB01 ctRNA, a transcript which consists of 54 nucleotides (nts), is encoded on the opposite strand from the copA/repB intergenic region and partially overlaps an atypical ribosome binding site (ARBS) for repB. The ARBS is integrated by the two underlined conserved regions: 5'-TTTTTGTNNNNTAANNNNNNNNNATG-3', and the ctRNA is complementary only to the 5' conserved sequence 5'-TTTTTGT-3'. This complementary sequence is located at a distance from the terminal loop of the ctRNA secondary structure. The ctRNA structure predicted by the mfold program suggests the possible generation of a terminal and an internal hairpin loop. The amount of in vitro translation product of repB mRNA was inversely proportional to the ctRNA concentration. Mutations in the terminal and internal hairpin loops of the ctRNA had inhibitory effects on its binding to the target mRNA. We propose that the intact structures of the terminal and internal hairpin loops, respectively, play important roles in forming the initial kissing and extending complexes between the ctRNA and target mRNA and that these regulate the copy number of this plasmid.

Keywords

Acknowledgement

Supported by : Ministry of Commerce, Industry and Energy (MOCIE)

References

  1. Bargonetti, J., Wang, P.-Z., and Novick, R.P. (1993). Measurement of gene expression by translational coupling: effect of copy mutations on pT181 initiator synthesis. EMBO J. 12, 3659-3667
  2. Bensing, B.A., and Dunny, G.M. (1993). Cloning and molecular analysis of genes affecting expression of binding substance, the recipient-encoded receptor(s) mediating mating aggregate formation in Enterococcus Faecalls. J. Bacteriol. 175, 7421-7429 https://doi.org/10.1128/jb.175.22.7421-7429.1993
  3. Brantl, S., and Wagner, E.G.-H. (2000). Antisense RNA mediated transcriptional attenuation: an in vitro study of plasmid pT181. Mol. Microbiol. 35, 1469-1482 https://doi.org/10.1046/j.1365-2958.2000.01813.x
  4. Brantl, S., and Wagner, E.G.-H. (2002). An antisense RNAmediated transcriptional attenuation mechanism functions in E. coli . J. Bacterial. 184, 2740-2747 https://doi.org/10.1128/JB.184.10.2740-2747.2002
  5. Carleton, S., Projan, S.J., Highlander, S.K., Moghazeh, S.M., and Novick, R.P. (1984). Control of pT181 replication II. Mutational analysis. EMBO J. 3, 2407-2414
  6. Costa, M., Sola, M., del Solar, G., Eritja, R., Hernandez-Arringa, A.M., Espinosa, M., Gomis-Ruth, F.X., and Coll, M. (2001). Plasmid transcriptional repressor CopG oligomerises to render helical superstructures unbound and in complexes with oligonucleotides. J. Mol. Biol. 310, 403-417 https://doi.org/10.1006/jmbi.2001.4760
  7. d'Aubenton-Carafa, Y., Brody, E., and Thermes, C. (1990). Prediction of Rho-independent Escherichia coli Acaa transcription terminators. A statistical analysis of their RNA stem-loop structures. J. Mol. Biol. 216, 835-858 https://doi.org/10.1016/S0022-2836(99)80005-9
  8. del Solar, G., and Espinosa, M. (1992). The copy number of plasmid pLS1 is regulated by two trans-acting plasmid products: the antisense RNAII and the repressor protein, RepA. Mol. Microbiol. 6, 83-94 https://doi.org/10.1111/j.1365-2958.1992.tb00840.x
  9. del Solar, G., and Espinosa, M. (2001). in vitro analysis of the terminator T (II) of the inhibitor antisense rna ll gene from plasmid pMV158. Plasmid 45, 75-87 https://doi.org/10.1006/plas.2000.1503
  10. del Solar, G.H., de la Campa, A.G., Perez-Martin, J., Choli, T., and Espinosa, M. (1989). Purification and characterization of RepA, a protein involved in the copy number control of plasmid pLS1. Nucleic Acids Res. 17, 2405-2420 https://doi.org/10.1093/nar/17.7.2405
  11. del Solar, G., Acebo, P., and Espinosa, M. (1995). Replication control of plasmid pLS1: efficient regulation of plasmid copy number is exerted by the combined action of two plasmid components, CopG and RNAII. Mol. Microbiol. 18, 913-924 https://doi.org/10.1111/j.1365-2958.1995.18050913.x
  12. del Solar, G., Acebo, P., and Espinosa, M. (1997). Replication control of plasmid pLS1: the antisense RNAII and the compact rnall region are involved in translational regulation of the initiator RepB synthesis. Mol. Microbiol. 23, 95-108 https://doi.org/10.1046/j.1365-2958.1997.1981561.x
  13. Dunny, G.M., Lee, L.N., and LeBlanc, D.J. (1991). Improved electroporation and cloning vector system for Gram-positive bacteria. Appl. Environ. Microbiol. 57, 1194-1201
  14. Franch, T., Petersen, M., Wagner, E.G.-H., Jacobsen, J.P., and Gerdes, K. (1999). Antisense RNA regulation in prokaryotes: Rapid RNA/RNA interaction facilitated by a general U-turn loop structure. J. Mol. Biol. 294, 1115-1125 https://doi.org/10.1006/jmbi.1999.3306
  15. Gomis-Ruth, F.X., Sola, M., Acebo, P., Parraga, A., Guasch, A., Eritja, R., Gonzalez, A., Espinosa, M., del Solar, G., and Coll, M. (1998). The structure of plasmid-encoded transcriptional repressor CopG unliganded and bound to its operator. EMBO J. 17, 7404-7415 https://doi.org/10.1093/emboj/17.24.7404
  16. Gutell, R.R., Cannone, J.J., Konings, D., and Gautheret, D. (2000). Predicting U-turns in ribosomal RNA with comparative sequence analysis. J. Mol. Biol. 300, 791-803 https://doi.org/10.1006/jmbi.2000.3900
  17. Khan, A.A., Nawaz, M.S., Khan, S.A., and Steele, R. (2002). Detection and characterization of erythromycin-resistant methylase genes in Gram-positive bacteria isolated from poultry litter. Appl. Microbiol. Biotechnol. 59, 377-381 https://doi.org/10.1007/s00253-002-1013-9
  18. Kim, S.W., Jeong, E.J., Kang, H.S., Tak, J.I., Bang, W.Y., Heo, J.B., Jeong, J.Y., Yoon, G.M., Kang, H.Y., and Bahk, J.D. (2006). Role of RepB in the replication of plasmid pJB01 isolated from Enterococcus faecium JC1. Plasmid 55, 99-113 https://doi.org/10.1016/j.plasmid.2005.08.002
  19. Kim, S.W., Bae, Y.G., Pyo, S.N., and Rhee, D.K. (2007). Differential regulation of the genes of the Streptococcus pneumpniae dnaK operon by Ca++. Mol. Cells 23, 239-245
  20. Kittle, J.D., Simons, R.W., Lee, J., and Kleckner, N. (1989). Insertion sequence ISNM anti-sense pairing initiates by an interaction between the 5′ end of the target RNA and a loop in the antisense RNA. J. Mol. Biol. 210, 561-572 https://doi.org/10.1016/0022-2836(89)90132-0
  21. Koepsel, R.R., Murray, R.W., Rosenblum, W.D., and Khan, S.A. (1985). The replication initiator protein of plasmid pT181 has sequence- specific endonuclease and topoisomerase-like activities. Proc. Natl. Acad. Sci. USA 82, 6845-6849
  22. Kolb, F.A., Westhof, E., Ehresmann, C., Ehresmann, B., Wagner, E.G.-H., and Romby, P. (2001). Bulged residues promote the progression of a loop-loop interaction to a stable and inhibitory antisense-target RNA complex. Nucleic Acids Res. 29, 3145- 3153 https://doi.org/10.1093/nar/29.15.3145
  23. Kumar, C.C., and Novick, R.P. (1985). Plasmid pT181 replication is regulated by two countertranscripts. Proc. Natl. Acad. Sci. USA 82, 638-642
  24. Kwak, J.-H., and Weisblum, B. (1994). Regulation of plasmid pE194 replication: control of AceJeEec operon transcription by Cop and of repF translation by countertranscript RNA. J. Bacteriol. 176, 5044-5051 https://doi.org/10.1128/jb.176.16.5044-5051.1994
  25. Mathews, D.H., Sabina, J., Zuker, M., and Turner, D.H. (1999). Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288, 911-940 https://doi.org/10.1006/jmbi.1999.2700
  26. Mcpherson, M.J., Quirke, P., and TayloR, G.R. (1993). PCR, A Practical Approach. IRL Press, pp. 207-209
  27. Nordstrom, K. (1983). Control of plasmid replication. Plasmid 9, 1-7 https://doi.org/10.1016/0147-619X(83)90026-4
  28. Nordstrom, K., and Austin, S.J. (1989). Mechanisms that contribute to the stable segregation of plasmids. Annu. Rev. Genet. 23, 37- 69 https://doi.org/10.1146/annurev.ge.23.120189.000345
  29. Novick, R.P. (1987). Plasmid incompatibility. Microbiol. Rev. 51, 381-395
  30. Novick, R.P., Iordanescu, S., Projan, S.J., Kornblum, J., and Edelman, I. (1989). pT181 plasmid replication is regulated by a countertranscript driven transcriptional attenuator. Cell 59, 395-404 https://doi.org/10.1016/0092-8674(89)90300-0
  31. O'sullivan, D.J., and Klaenhammer, T.R. (1993). Rapid mini-prep isolation of high-quality plasmid DNA from Lactococcus and Lactocobacillus spp. Appl. Environ. Microbiol. 59, 2730-2733
  32. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular cloning, a laboratory manual. 2nd eds. In Cold Spring Harbor Laboratory, (NY, USA: Cold Spring Harbor), pp. 1.25-28, 1.38- 41
  33. Thisted, T., Sorensen, N.S., Wagner, E.G.H., and Gerdes, K. (1994). Mechanism of post-segregational killing: Sok antisense RNA interacts with Uca mRNA via its 5′-end singlestranded leader and competes with the 3′-end of Uca mRNA for binding to the aca translational initiation region. EMBO J. 13, 1960-1968
  34. van der Vossen, J.M., Kok, J., and Venema, G. (1985). Construction of cloning, promoter-screening, and terminator-screening shuttle vectors for Bacillus and Streptococcus. Appl. Environ. Microbiol. 50, 540-542
  35. Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 1-10 https://doi.org/10.1093/nar/gkg120