DOI QR코드

DOI QR Code

Artificial antisense RNAs silence lacZ in E. coli by decreasing target mRNA concentration

  • Published : 2008.08.31

Abstract

Antisense RNA molecules are powerful tools for controlling the expression of specific genes but their use in prokaryotes has been limited by their unpredictable antisense effectiveness. Moreover, appreciation of the molecular mechanisms associated with silencing in bacteria is still restricted. Here we report our attempts to define an effective antisense strategy in E. coli, and to dissect the observed silencing process. Antisense constructs complementary to different regions of lacZ were investigated, and silencing was observed exclusively upon expression of antisense RNA hybridising the 5'UTR of lac messenger. The level of lacZ mRNA was reduced upon expression of this antisense construct, and the silencing competence was found to be closely associated with its stability. These observations may help in the design of antisense molecules directed against prokaryotic genes.

Keywords

References

  1. Link, A. J., Phillips, D. and Church, G. M. (1997) Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization. J. Bacteriol. 179, 6228-6237. https://doi.org/10.1128/jb.179.20.6228-6237.1997
  2. Yu, D., Ellis, H. M., Lee, E., Jenkins, N. A., Copeland, N. G. and Court, D. L. (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 97, 5978-5983. https://doi.org/10.1073/pnas.100127597
  3. Zhilina, Z. V., Ziemba, A. J. and Ebbinghaus, S. W. (2005) Peptide nucleic acids conjugates: synthesis, properties and applications. Curr. Top. Med. Chem. 12, 1119-1131.
  4. Ji, Y., Zhang, B., Van Horn, S. F., Warren, P., Woodnutt, G., Burnham, M. K. R. and Rosenberg, M. (2001) Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA. Science 293, 2266-2269. https://doi.org/10.1126/science.1063566
  5. Gottesman, S. (2005) Micros for microbes: non-coding regulatory RNAs in bacteria. Trends Genet. 21, 399-404. https://doi.org/10.1016/j.tig.2005.05.008
  6. Coleman, J., Green, P. J. and Inouye, M. (1984) The use of RNAs complementary to specific mRNAs to regulate the expression of individual bacterial genes. Cell 37, 429-436. https://doi.org/10.1016/0092-8674(84)90373-8
  7. Nakashima, N., Tamura, T. and Good, L. (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli. Nucleic Acids Res. 34, e138. https://doi.org/10.1093/nar/gkl697
  8. Makarova, K. S., Grishin, N. V., Shabalina, S. A., Wolf, Y. I. and Koonin, E. V. (2006) A putative RNA-interference- based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanism of action. Biology Direct 1, 7. (doi: 10.1186/1745-6150-1-7).
  9. Pestka, S., Daugherty, B. L., Jung, V., Hotta, K. and Pestka, R. K. (1984) Anti-mRNA: specific inhibition of translation of single mRNA molecules. Proc. Natl. Acad. Sci. U.S.A. 81, 7525-7528. https://doi.org/10.1073/pnas.81.23.7525
  10. Ellison, M. J., Kelleher III, R. J. and Rich, A. (1985) Thermal regulation of $\beta$-galactosidase synthesis using antisense RNA directed against the coding portion of the mRNA. J. Biol. Chem. 260, 9085-9087.
  11. Stefan, A., Reggiani, L., Cianchetta, S., Radeghieri, A., Gonzalez Vara y Rodriguez, A. and Hochkoeppler, A. (2003) Silencing of the gene coding for the $\varepsilon$ subunit of DNA polymerase III slows down the growth rate of Escherichia coli populations. FEBS Letts. 546, 295-299. https://doi.org/10.1016/S0014-5793(03)00604-5
  12. Stefan, A., Tabler, M. and Hochkoeppler, A. (2007) Efficient silencing of the gene coding for the $\varepsilon$ subunit of DNA polymerase III in Escherichia coli is triggered by antisense RNAs featuring stability in vivo. FEMS Microbiol. Letts. 270, 277-283. https://doi.org/10.1111/j.1574-6968.2007.00679.x
  13. Brown, J. L., Brown, D. M. and Zabin, I. (1967) Thiogalactoside transacetylase. Physical and chemical studies of subunit structure. J. Biol. Chem. 242, 4254-4258.
  14. Li, Y. and Altman, S. (2004) Polarity effects in the lactose operon of Escherichia coli. J. Mol. Biol. 339, 31-39. https://doi.org/10.1016/j.jmb.2004.03.041
  15. Dryselius, R., Nikravesh, A., Kulytè, A., Goh, S. and Good, L. (2006) Variable coordination of cotranscribed genes in Escherichia coli following antisense repression. BMC Microbiology 6, 97-106. https://doi.org/10.1186/1471-2180-6-97
  16. Radeghieri, A., Bonoli, M., Parmeggiani, F. and Hochkoeppler A. (2007) Tyrosine83 is essential for the activity of E. coli galactoside transacetylase. Biochim. Biophys. Acta. 1774, 243-248. https://doi.org/10.1016/j.bbapap.2006.11.013
  17. Gultyaev, A. P., van Batenburg F. H. D. and Pleij, C. W. A. (1995) The computer simulation of RNA folding pathways using a genetic algorithm. J. Mol. Biol. 250, 37-51. https://doi.org/10.1006/jmbi.1995.0356
  18. De Rijk, P. and De Wachter, R. (1997) RnaViz, a program for the visualization of RNA secondary structure. Nucleic Acids Res. 25, 4679-4684. https://doi.org/10.1093/nar/25.22.4679
  19. Miller, J. H. (1992) A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, New York, U.S.A.

Cited by

  1. Development of Design Rules for Reliable Antisense RNA Behavior in E. coli vol.5, pp.12, 2016, https://doi.org/10.1021/acssynbio.6b00036
  2. Shine-Dalgarno sequence enhances the efficiency of lacZ repression by artificial anti-lac antisense RNAs in Escherichia coli vol.110, pp.5, 2010, https://doi.org/10.1016/j.jbiosc.2010.05.012
  3. On-off controllable RNA hybrid expression vector for yeast three-hybrid system vol.43, pp.2, 2010, https://doi.org/10.5483/BMBRep.2010.43.2.110