Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Distinct mechanisms coordinate transcription and translation under carbon and nitrogen starvation in Escherichia coli

Nature Microbiology volume 3, pages 741–748 (2018)Cite this article

Subjects

Abstract

Bacteria adapt to environmental stress by producing proteins that provide stress protection. However, stress can severely perturb the kinetics of gene expression, disrupting protein production. Here, we characterized how Escherichia coli mitigates such perturbations under nutrient stress through the kinetic coordination of transcription and translation. We observed that, when translation became limiting under nitrogen starvation, transcription elongation slowed accordingly. This slowdown was mediated by (p)ppGpp, the alarmone whose primary role is thought to be promoter regulation. This kinetic coordination by (p)ppGpp was critical for the robust synthesis of gene products. Surprisingly, under carbon starvation, (p)ppGpp was dispensable for robust synthesis. Characterization of the underlying kinetics revealed that under carbon starvation, transcription became limiting, and translation aided transcription elongation. This mechanism naturally coordinated transcription with translation, alleviating the need for (p)ppGpp as a mediator. These contrasting mechanisms for coordination resulted in the condition-dependent effects of (p)ppGpp on global protein synthesis and starvation survival. Our findings reveal a kinetic aspect of gene expression plasticity, establishing (p)ppGpp as a condition-dependent global effector of gene expression.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Effects of C- and N starvation on gene expression kinetics.
Fig. 2: Characterization of transcription elongation and translation elongation.
Fig. 3: Contrasting effects of (p)ppGpp on transcription and translation kinetics under N- and C starvation.
Fig. 4: Condition-dependent effects of (p)ppGpp on global protein synthesis activity and cell survival.

Similar content being viewed by others

References

  1. Browning, D. F. & Busby, S. J. W. Local and global regulation of transcription initiation in bacteria. Nat. Rev. Micro 14, 638–650 (2016).

    Article  CAS  Google Scholar 

  2. Schellhorn, H. E. & Hassan, H. M. Transcriptional regulation of katE in Escherichia coli K-12. J. Bacteriol 170, 4286–4292 (1988).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Santos, J. M., Lobo, M., Matos, A. P. A., De Pedro, M. A. & Arraiano, C. M. The gene bolA regulates dacA (PBP5), dacC (PBP6) and ampC (AmpC), promoting normal morphology in Escherichia coli. Mol. Microbiol 45, 1729–1740 (2002).

    Article  PubMed  CAS  Google Scholar 

  4. Mengin-Lecreulx, D. & van Heijenoort, J. Effect of growth conditions on peptidoglycan content and cytoplasmic steps of its biosynthesis in Escherichia coli. J. Bacteriol. 163, 208–212 (1985).

    PubMed  PubMed Central  CAS  Google Scholar 

  5. Matin, A., Auger, E. A., Blum, P. H., a. & Schultz, J. E. Genetic basis of starvation survival in nondifferentiating bacteria. Annu. Rev. Microbiol. 43, 293–314 (1989).

    Article  PubMed  CAS  Google Scholar 

  6. Mellies, J., Wise, A. & Villarejo, M. Two different Escherichia coli proP promoters respond to osmotic and growth phase signals. J. Bacteriol. 177, 144–151 (1995).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Yoshida, H., Yamamoto, H., Uchiumi, T. & Wada, A. RMF inactivates ribosomes by covering the peptidyl transferase centre and entrance of peptide exit tunnel. Genes Cells 9, 271–278 (2004).

    Article  PubMed  CAS  Google Scholar 

  8. Cagliero, C. & Jin, D. J. Dissociation and re-association of RNA polymerase with DNA during osmotic stress response in Escherichia coli. Nucleic Acids Res. 41, 315–326 (2013).

    Article  PubMed  CAS  Google Scholar 

  9. McGary, K. & Nudler, E. RNA polymerase and the ribosome: the close relationship. Curr. Opin. Microbiol. 16, 112–117 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Ray-Soni, A., Bellecourt, M. J. & Landick, R. Mechanisms of bacterial transcription termination: all good things must end. Annu. Rev. Biochem. 85, 319–347 (2016).

    Article  PubMed  CAS  Google Scholar 

  11. Morita, R. Y. Bacteria in Oligotrophic Environments: Starvation-Survival Lifestyle (Chapman & Hall, London, 1997).

  12. Reeve, C. A., Amy, P. S. & Matin, A. Role of protein synthesis in the survival of carbon-starved Escherichia coli K-12. J. Bacteriol. 160, 1041–1046 (1984).

    PubMed  PubMed Central  CAS  Google Scholar 

  13. Houser, J. R. et al. Controlled measurement and comparative analysis of cellular components in E. coli reveals broad regulatory changes in response to glucose starvation. PLoS Comput. Biol. 11, e1004400 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Phaiboun, A., Zhang, Y., Park, B. & Kim, M. Survival kinetics of starving bacteria is biphasic and density-dependent. PLoS Comput. Biol. 11, e1004198 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Iyer, S., Park, B. R. & Kim, M. Absolute quantitative measurement of transcriptional kinetic parameters in vivo. Nucleic Acids Res. 44, e142 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Garcia, H. G., Lee, H. J., Boedicker, J. Q. & Phillips, R. Comparison and calibration of different reporters for quantitative analysis of gene expression. Biophys. J. 101, 535–544 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Bremer, H. & Dennis, P. Modulation of chemical composition and other parameters of the cell at different exponential growth rates. EcoSal Plus https://doi.org/10.1128/ecosal.5.2.3 (2008).

  18. Proshkin, S., Rahmouni, A. R., Mironov, A. & Nudler, E. Cooperation between translating ribosomes and RNA polymerase in transcription elongation. Science 328, 504–508 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Kepes, A. Transcription and translation in the lactose operon of Escherichia coli studied by in vivo kinetics. Progress. Biophys. Mol. Biol. 19, 199–236 (1969).

    Article  CAS  Google Scholar 

  20. Schleif, R., Hess, W., Finkelstein, S. & Ellis, D. Induction kinetics of the l-arabinose operon of Escherichia coli. J. Bacteriol. 115, 9–14 (1973).

    PubMed  PubMed Central  CAS  Google Scholar 

  21. Jin, D. J., Burgess, R. R., Richardson, J. P. & Gross, C. A. Termination efficiency at rho-dependent terminators depends on kinetic coupling between RNA polymerase and rho. Proc. Natl Acad. Sci. USA 89, 1453–1457 (1992).

    Article  PubMed  CAS  Google Scholar 

  22. Dai, X. et al. Reduction of translating ribosomes enables Escherichia coli to maintain elongation rates during slow growth. Nat. Microbiol 2, 16231 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Komissarova, N. & Kashlev, M. RNA polymerase switches between inactivated and activated states by translocating back and forth along the DNA and the RNA. J. Biol. Chem. 272, 15329–15338 (1997).

    Article  PubMed  CAS  Google Scholar 

  24. Nudler, E., Mustaev, A., Goldfarb, A. & Lukhtanov, E. The RNA–DNA hybrid maintains the register of transcription by preventing backtracking of RNA polymerase. Cell 89, 33–41 (1997).

    Article  PubMed  CAS  Google Scholar 

  25. Mamata, S. & Stefan, K. Backtracking dynamics of RNA polymerase: pausing and error correction. J. Phys. Condens. Matter 25, 374104 (2013).

    Article  CAS  Google Scholar 

  26. Steitz, T. A. A structural understanding of the dynamic ribosome machine. Nat. Rev. Mol. Cell Biol. 9, 242–253 (2008).

    Article  PubMed  CAS  Google Scholar 

  27. Zhang, Y. et al. DksA guards elongating RNA polymerase against ribosome-stalling-induced arrest. Mol. Cell 53, 766–778 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Potrykus, K. & Cashel, M. (p)ppGpp: still magical? Annu. Rev. Microbiol. 62, 35–51 (2008).

    Article  PubMed  CAS  Google Scholar 

  29. Villadsen, I. S. & Michelsen, O. Regulation of PRPP and nucleoside tri and tetraphosphate pools in Escherichia coli under conditions of nitrogen starvation. J. Bacteriol. 130, 136–143 (1977).

    PubMed  PubMed Central  CAS  Google Scholar 

  30. Metzger, S., Schreiber, G., Aizenman, E., Cashel, M. & Glaser, G. Characterization of the relA1 mutation and a comparison of relA1 with new relA null alleles in Escherichia coli. J. Biol. Chem. 264, 21146–21152 (1989).

    PubMed  CAS  Google Scholar 

  31. Gentry, D. R. & Cashel, M. Mutational analysis of the Escherichia coli spoT gene identifies distinct but overlapping regions involved in ppGpp synthesis and degradation. Mol. Microbiol. 19, 1373–1384 (1996).

  32. Ryals, J., Little, R. & Bremer, H. Control of rRNA and tRNA syntheses in Escherichia coli by guanosine tetraphosphate. J. Bacteriol. 151, 1261–1268 (1982).

    PubMed  PubMed Central  CAS  Google Scholar 

  33. Cashel, M. & Gallant, J. Two compounds implicated in the function of the RC gene of Escherichia coli. Nature 221, 838–841 (1969).

    Article  PubMed  CAS  Google Scholar 

  34. Spira, B., Silberstein, N. & Yagil, E. Guanosine 3’,5’-bispyrophosphate (ppGpp) synthesis in cells of Escherichia coli starved for Pi. J. Bacteriol. 177, 4053–4058 (1995).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Kingston, R. E., Nierman, W. C. & Chamberlin, M. J. A direct effect of guanosine tetraphosphate on pausing of Escherichia coli RNA polymerase during RNA chain elongation. J. Biol. Chem. 256, 2787–2797 (1981).

  36. Kingston, R. E. & Chamberlin, M. J. Pausing and attenuation of in vitro transcription in the rrnB operon of E. coli. Cell 27, 523–531 (1981).

    Article  PubMed  CAS  Google Scholar 

  37. Krohn, M. & Wagner, R. Transcriptional pausing of RNA polymerase in the presence of guanosine tetraphosphate depends on the promoter and gene sequence. J. Biol. Chem. 271, 23884–23894 (1996).

    Article  PubMed  CAS  Google Scholar 

  38. Vogel, U., Sørensen, M., Pedersen, S., Jensen, K. F. & Kilstrup, M. Decreasing transcription elongation rate in Escherichia coli exposed to amino acid starvation. Mol. Microbiol. 6, 2191–2200 (1992).

  39. Sørensen, M. A., Jensen, K. F. & Pedersen, S. High concentrations of ppGpp decrease the RNA chain growth rate. J. Mol. Biol. 236, 441–454 (1994).

    Article  PubMed  Google Scholar 

  40. van Ooyen, A. J. J., Gruber, M. & Jørgensen, P. The mechanism of action of ppGpp on rRNA synthesis in vitro. Cell 8, 123–128 (1976).

    Article  PubMed  Google Scholar 

  41. Barker, M. M., Gaal, T., Josaitis, C. A. & Gourse, R. L. Mechanism of regulation of transcription initiation by ppGpp. I. Effects of ppGpp on transcription initiation in vivo and in vitro. J. Mol. Biol. 305, 673–688 (2001).

    Article  PubMed  CAS  Google Scholar 

  42. Travers, A. A. Promoter sequence for stringent control of bacterial ribonucleic acid synthesis. J. Bacteriol. 141, 973–976 (1980).

    PubMed  PubMed Central  CAS  Google Scholar 

  43. Ross, W. et al. ppGpp binding to a site at the RNAP-DksA interface accounts for its dramatic effects on transcription initiation during the stringent response. Mol. Cell 62, 811–823 (2016).

  44. Roghanian, M., Zenkin, N. & Yuzenkova, Y. Bacterial global regulators DksA/ppGpp increase fidelity of transcription. Nucleic Acids Res. 43, 1529–1536 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Svitil, A. L., Cashel, M. & Zyskind, J. W. Guanosine tetraphosphate inhibits protein synthesis in vivo. A possible protective mechanism for starvation stress in Escherichia coli. J. Biol. Chem. 268, 2307–2311 (1993).

    PubMed  CAS  Google Scholar 

  46. Xiao, H. et al. Residual guanosine 3',5'-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations. J. Biol. Chem. 266, 5980–5990 (1991).

  47. Harinarayanan, R., Murphy, H. & Cashel, M. Synthetic growth phenotypes of Escherichia coli lacking ppGpp and transketolase A (tktA) are due to ppGpp-mediated transcriptional regulation of tktB. Mol. Microbiol. 69, 882–894 (2008).

  48. Haugen, S. P., Ross, W. & Gourse, R. L. Advances in bacterial promoter recognition and its control by factors that do not bind DNA. Nat. Rev. Micro. 6, 507–519 (2008).

    Article  CAS  Google Scholar 

  49. Durfee, T., Hansen, A.-M., Zhi, H., Blattner, F. R. & Jin, D. J. Transcription profiling of the stringent response in Escherichia coli. J. Bacteriol. 190, 1084–1096 (2008).

    Article  PubMed  CAS  Google Scholar 

  50. Brown, D. R., Barton, G., Pan, Z., Buck, M. & Wigneshweraraj, S. Nitrogen stress response and stringent response are coupled in Escherichia coli. Nat. Commun. 5, 4115 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Soupene, E. et al. Physiological studies of Escherichia coli strain MG1655: growth defects and apparent cross-regulation of gene expression. J. Bacteriol. 185, 5611–5626 (2003).

  52. Lyons, E., Freeling, M., Kustu, S. & Inwood, W. Using genomic sequencing for classical genetics in E. coli K12. PLoS ONE 6, e16717 (2011).

  53. Brown, S. D. & Jun, S. Complete genome sequence of Escherichia coli NCM3722. Genome Announc. 3 (2015).

  54. Csonka, L. N., Ikeda, T. P., Fletcher, S. A. & Kustu, S. The accumulation of glutamate is necessary for optimal growth of Salmonella typhimurium in media of high osmolality but not induction of the proU operon. J. Bacteriol. 176, 6324–6333 (1994).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Kim, M., Zhang, Z. G., Okano, H., Yan, D. L., Groisman, A. & Hwa, T. Need-based activation of ammonium uptake in Escherichia coli. Mol. Syst. Biol. 8, 616 (2012).

    PubMed  PubMed Central  Google Scholar 

  56. Novick, A. & Weiner, M. Enzyme induction as an all-or-none phenomenon. Proc. Natl Acad. Sci. USA 43, 553–566 (1957).

    Article  PubMed  CAS  Google Scholar 

  57. Kuhlman, T., Zhang, Z., Saier, M. H. Jr & Hwa, T. Combinatorial transcriptional control of the lactose operon of Escherichia coli. Proc. Natl Acad. Sci. USA 104, 6043–6048 (2007).

  58. Miller, J. H. Experiments in Molecular Genetics (Cold Spring Harbor Laboratory Press, New York, 1972).

  59. Dieterich, D. C., Link, A. J., Graumann, J., Tirrell, D. A. & Schuman, E. M. Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc. Natl Acad. Sci. USA 103, 9482–9487 (2006).

  60. Beatty, K. E. et al. Live-cell imaging of cellular proteins by a strain-promoted azide–alkyne cycloaddition. ChemBioChem 11, 2092–2095 (2010).

  61. Hatzenpichler, R. et al. In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry. Environ. Microbiol. 16, 2568–2590 (2014).

  62. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).

    Article  PubMed  CAS  Google Scholar 

  63. Merril, C. R. Gel-staining techniques. Methods Enzymol. 182, 477–488 (1990).

Download references

Acknowledgements

We thank M. Cashel, R.L. Gourse and J. Wang for helpful discussions and kindly sharing strains with us. This work was funded by Research Corporation for Science Advancement (24097) and the Human Frontier Science Program (RGY0072/2015).

Author information

Authors and Affiliations

  1. Department of Physics, Emory University, Atlanta, GA, USA

    Sukanya Iyer, Dai Le, Bo Ryoung Park & Minsu Kim

  2. Graduate Division of Biological and Biomedical Sciences, Emory University, Atlanta, GA, USA

    Minsu Kim

  3. Emory Antibiotic Resistance Center, Emory University, Atlanta, GA, USA

    Minsu Kim

Authors
  1. Sukanya Iyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dai Le
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bo Ryoung Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Minsu Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.I., D.L., B.R.P. and M.K. designed the study. S.I., D.L. and B.R.P. performed experiments. S.I. D.K. and B.R.P. analysed the data. M.K. wrote the manuscript.

Corresponding author

Correspondence to Minsu Kim.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Methods, Supplementary Figures 1–22, Supplementary Tables 1–5, Supplementary References.

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Iyer, S., Le, D., Park, B.R. et al. Distinct mechanisms coordinate transcription and translation under carbon and nitrogen starvation in Escherichia coli. Nat Microbiol 3, 741–748 (2018). https://doi.org/10.1038/s41564-018-0161-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41564-018-0161-3

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing