A Proteomic Approach to Study msDNA Function in Escherichia coli

  • Jeong, Mi-Ae (Department of Bioinformatics and Life Science Soongsil University) ;
  • Lim, Dongbin (Department of Bioinformatics and Life Science Soongsil University)
  • Published : 2004.09.01

Abstract

Retron is a prokaryotic genetic element that produces multicopy single-stranded DNA covalently linked to RNA (msDNA) by a reverse transcriptase. It was found that cells producing a large amount of msDNA, rather than those that did not, showed a higher rate of mutation. In order to understand the molecular mechanism connecting msDNA production to the high mutation rate the protein patterns were compared by two dimensional gel electrophoresis. Ten proteins were found to be differentially expressed at levels more than three fold greater in cells with than without msDNA, nine of which were identified by MALDI TOF MS. Eight of the nine identified proteins were repressed in msDNA-producing cells and, surprisingly, most were proteins functioning in the dissimilation of various carbon sources. One protein was induced four fold greater in the msDNA producing cells and was identified as a 30S ribosomal protein S2 involved in the regulation of translation. The molecular mechanism underlying the elevated mutation in msDNA-producing cell still remains elusive.

Keywords

References

  1. Blum, H., H. Beier, and H.J. Gross. 1987. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93-99
  2. Choy, H.E., S.W Park, P. Parrack, and S. Adhya. 1995. Transcription regulation by inflexibility of promoter DNA in a looped complex. Proc. Natl. Acad. Sci. USA 92, 7327-7331
  3. Cupples, C.G., M. Cabrera, C. Cruz, and J.H. Miller. 1990. A set of lacZ mutations in Escherichia coli that allow rapid detection of specific frameshit mutations. Genetics 125, 275-280
  4. Furuichi, T., S. Inouye, and M. Inouye. 1987. Biosynthesis and structure of stable branched RNA covalently linked to the 5' end of multicopy single-stranded DNA of Stigmatella aurantiaca. Cell 48, 55-62
  5. Hagewood, B.T., Y.L. Ganduri, and P. Datta. 1994. Functional analysis of the tdcABC promoter of Escherichia coli: roles of TdcA and TdcR. J. Bacteriol. 176, 6214-6220
  6. Hesslinger, C., S.A. Fairhurst, and G. Sawers. 1998. Novel keto acid formate-lyase and propionate kinase enzymes are components of an anaerobic pathway in Escherichia coli that degrades L-threonine to propionate. Mol. Microbiol. 27, 477-492
  7. Jurgen, B., R. Hanschke, M. Sarvas, M. Hecker, and T. Schweder. 2001. Proteome and transcriptome based analysis of Bacillus subtilis cells overproducing an insoluble heterologous protein. Appl. Microbiol. Biotechnol. 55, 326-332
  8. Lee, K., D. Bae, and D. Lim. 2002. Evaluation of parameters in peptide mass fingerprinting for protein identification by MALDI-TOF mass spectrometry. Mol. Cells. 13, 175-184
  9. Lia, G., D. Bensimon, V. Croquette, J.F. Allemand, D. Dunlap, D.E. Lewis, S. Adhya, and L. Finzi. 2003. Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)- mediated DNA looping. Proc. Natl. Acad. Sci. USA 100, 11373-11377
  10. Lim, D. and W.K. Maas. 1989. Reverse transcriptase-dependent synthesis of a covalently linked, branched DNA-RNA compound in E. coli B. Cell 56, 891-904
  11. Lim, D. 1991. Structure of two retrons of Escherichia coli and their common chromosomal insertion site. Mol. Microbiol. 5, 1863-1872
  12. Maas, W. K., C. Wang, T. Lima, G. Zubay, and D. Lim. 1994. Multicopy single-stranded DNAs with mismatched base pairs are mutagenic in Escherichia coli. Mol. Microbiol. 14, 437-441
  13. Moll, I., S. Grill, A. Grundling, and U. Blasi. 2002. Effects of ribosomal proteins S1, S2 and the DeaD/CsdA DEAD-box helicase on translation of leaderless and canonical mRNAs in Escherichia coli. Mol. Microbiol. 44, 1387-1396
  14. Nobelmann, B. and J.W. Lengeler. 1995. Sequence of the gat operon for galactitol utilization from a wild-type strain EC3132 of Escherichia coli. Biochim. Biophys. Acta 1262, 69-72
  15. Nobelmann, B. and J.W. Lengeler. 1996. Molecular analysis of the gat genes from Escherichia coli and of their roles in galactitol transport and metabolism. J. Bacteriol. 178, 6790-6795
  16. Ozbudak, E.M., M. Thattai H.N. Lim, B.I. Shraiman, and A. van Oudenaarden. 2004. Multistability in the lactose utilization network of Escherichia coli. Nature 427, 737-740
  17. Shean, C.S. and M.E. Gottesman 1992. Translation of the prophage lambda cl transcript. Cell 70, 513-522
  18. Temin, H. M. 1989. Retrons in bacteria. Nature 339, 254-255
  19. Truniger, V. and W. Boos. 1994. Mapping and cloning of gldA, the structural gene of the Escherichia coli glycerol dehydrogenase. J. Bacteriol. 176, 1796-1800
  20. Weickert, M.J. and S. Adhya. 1993. The galactose regulon of Escherichia coli. Mol. Microbiol. 10, 245-251