Disruption of transcription–translation coordination in Escherichia coli leads to premature transcriptional termination

Abstract

Tight coordination between transcription and translation is crucial to maintaining the integrity of gene expression in bacteria, yet how bacteria manage to coordinate these two processes remains unclear. Possible direct physical coupling between the RNA polymerase and ribosome has been thoroughly investigated in recent years. Here, we quantitatively characterize the transcriptional kinetics of Escherichia coli under different growth conditions. Transcriptional and translational elongation remain coordinated under various nutrient conditions, as previously reported. However, transcriptional elongation was not affected under antibiotics that slowed down translational elongation. This result was also found by introducing nonsense mutation that completely dissociated transcription from translation. Our data thus provide direct evidence that translation is not required to maintain the speed of transcriptional elongation. In cases where transcription and translation are dissociated, our study provides quantitative characterization of the resulting process of premature transcriptional termination (PTT). PTT-mediated polarity caused by translation-targeting antibiotics substantially affected the coordinated expression of genes in several long operons, contributing to the key physiological effects of these antibiotics. Our results also suggest a model in which the coordination between transcriptional and translational elongation under normal growth conditions is implemented by guanosine tetraphosphate.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: RT–qPCR-based characterization of transcriptional elongation speed.
Fig. 2: Transcriptional elongation under conditions of reduced translational elongation induced by FA.
Fig. 3: lacZ mRNA transcriptional induction kinetics following nonsense mutation and antibiotic treatment.
Fig. 4: Expression polarity of the r-protein operon under antibiotic treatment.
Fig. 5: Effect of ppGpp on transcriptional elongation.
Fig. 6: Coordination of transcriptional and translational elongation under nutrient limitation.

Data availability

The key data that support the findings of this study are summarized in the Supplementary tables. Other details are available from the corresponding author upon request.

References

  1. 1.

    Gowrishankar, J. & Harinarayanan, R. Why is transcription coupled to translation in bacteria? Mol. Microbiol. 54, 598–603 (2004).

    CAS  PubMed  Google Scholar 

  2. 2.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Vogel, U. & Jensen, K. F. The RNA chain elongation rate in Escherichia coli depends on the growth rate. J. Bacteriol. 176, 2807–2813 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Newton, W. A., Beckwith, J. R., Zipser, D. & Brenner, S. Nonsense mutants and polarity in the lac operon of Escherichia coli. J. Mol. Biol. 14, 290–296 (1965).

    CAS  PubMed  Google Scholar 

  6. 6.

    Elgamal, S., Artsimovitch, I. & Ibba, M. Maintenance of transcription–translation coupling by elongation factor P. mBio 7, e01373–16 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Adhya, S. & Gottesman, M. Control of transcription termination. Annu. Rev. Biochem. 47, 967–996 (1978).

    CAS  PubMed  Google Scholar 

  8. 8.

    Richardson, J. P. Preventing the synthesis of unused transcripts by Rho factor. Cell 64, 1047–1049 (1991).

    CAS  PubMed  Google Scholar 

  9. 9.

    Kohler, R., Mooney, R. A., Mills, D. J., Landick, R. & Cramer, P. Architecture of a transcribing-translating expressome. Science 356, 194–197 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Miller, O. L. Jr., Hamkalo, B. A. & Thomas, C. A. Jr. Visualization of bacterial genes in action. Science 169, 392–395 (1970).

    PubMed  Google Scholar 

  11. 11.

    Demo, G. et al. Structure of RNA polymerase bound to ribosomal 30S subunit. eLife 6, e28560 (2017).

    PubMed  PubMed Central  Google Scholar 

  12. 12.

    Fan, H. et al. Transcription–translation coupling: direct interactions of RNA polymerase with ribosomes and ribosomal subunits. Nucleic Acids Res. 45, 11043–11055 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Burmann, B. M. et al. A NusE:NusG complex links transcription and translation. Science 328, 501–504 (2010).

    CAS  PubMed  Google Scholar 

  14. 14.

    Saxena, S. et al. Escherichia coli transcription factor NusG binds to 70S ribosomes. Mol. Microbiol. 108, 495–504 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Burmann, B. M. et al. An alpha helix to beta barrel domain switch transforms the transcription factor RfaH into a translation factor. Cell 150, 291–303 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Chen, M. & Fredrick, K. Measures of single- versus multiple-round translation argue against a mechanism to ensure coupling of transcription and translation. Proc. Natl Acad. Sci. USA 115, 10774–10779 (2018).

    CAS  PubMed  Google Scholar 

  17. 17.

    Bremer, H. & Dennis, P. P. in Escherichia coli and Salmonella 2nd edn (ed. Neidhardt, F. C.) 1553–1569 (American Society of Microbiology, 1996).

  18. 18.

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

    PubMed  PubMed Central  Google Scholar 

  19. 19.

    Dai, X. et al. Slowdown of translational elongation in Escherichia coli under hyperosmotic stress. mBio 9, e02375–18 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

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

    PubMed  PubMed Central  Google Scholar 

  21. 21.

    Zhu, M., Dai, X. & Wang, Y. P. Real time determination of bacterial in vivo ribosome translation elongation speed based on LacZα complementation system. Nucleic Acids Res. 44, e155 (2016).

    PubMed  PubMed Central  Google Scholar 

  22. 22.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Bennett, P. M. & Maaloe, O. The effects of fusidic acid on growth, ribosome synthesis and RNA metabolism in Escherichia coli. J. Mol. Biol. 90, 541–561 (1974).

    CAS  PubMed  Google Scholar 

  24. 24.

    Seo, H. S. et al. EF-G-dependent GTPase on the ribosome. Conformational change and fusidic acid inhibition. Biochemistry 45, 2504–2514 (2006).

    CAS  PubMed  Google Scholar 

  25. 25.

    Richardson, L. V. & Richardson, J. P. Rho-dependent termination of transcription is governed primarily by the upstream Rho utilization (rut) sequences of a terminator. J. Biol. Chem. 271, 21597–21603 (1996).

    CAS  PubMed  Google Scholar 

  26. 26.

    Graham, J. E. & Richardson, J. P. rut sites in the nascent transcript mediate Rho-dependent transcription termination in vivo. J. Biol. Chem. 273, 20764–20769 (1998).

    CAS  PubMed  Google Scholar 

  27. 27.

    Ruteshouser, E. C. & Richardson, J. P. Identification and characterization of transcription termination sites in the Escherichia coli lacZ gene. J. Mol. Biol. 208, 23–43 (1989).

    CAS  PubMed  Google Scholar 

  28. 28.

    Harvey, R. J. & Koch, A. L. How partially inhibitory concentrations of chloramphenicol affect the growth of Escherichia coli. Antimicrob. Agents Chemother. 18, 323–337 (1980).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Maguire, B. A. Inhibition of bacterial ribosome assembly: a suitable drug target? Microbiol. Mol. Biol. Rev. 73, 22–35 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Hui, S. et al. Quantitative proteomic analysis reveals a simple strategy of global resource allocation in bacteria. Mol. Syst. Biol. 11, 784 (2015).

    PubMed  PubMed Central  Google Scholar 

  31. 31.

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

    CAS  PubMed  Google Scholar 

  32. 32.

    Vogel, U., Sorensen, 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).

    CAS  PubMed  Google Scholar 

  33. 33.

    Vogel, U. & Jensen, K. F. Effects of guanosine 3′,5′-bisdiphosphate (ppGpp) on rate of transcription elongation in isoleucine-starved Escherichia coli. J. Biol. Chem. 269, 16236–16241 (1994).

    CAS  PubMed  Google Scholar 

  34. 34.

    Iyer, S., Le, D., Park, B. R. & Kim, M. Distinct mechanisms coordinate transcription and translation under carbon and nitrogen starvation in Escherichia coli. Nat. Microbiol. 3, 741 (2018).

    CAS  PubMed  Google Scholar 

  35. 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).

    CAS  PubMed  Google Scholar 

  36. 36.

    Furman, R., Sevostyanova, A. & Artsimovitch, I. Transcription initiation factor DksA has diverse effects on RNA chain elongation. Nucleic Acids Res. 40, 3392–3402 (2012).

    CAS  PubMed  Google Scholar 

  37. 37.

    Klumpp, S., Zhang, Z. & Hwa, T. Growth rate-dependent global effects on gene expression in bacteria. Cell 139, 1366–1375 (2009).

    PubMed  PubMed Central  Google Scholar 

  38. 38.

    Schreiber, G. et al. Overexpression of the relA gene in Escherichia coli. J. Biol. Chem. 266, 3760–3767 (1991).

    CAS  PubMed  Google Scholar 

  39. 39.

    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).

    CAS  PubMed  Google Scholar 

  40. 40.

    Hernandez, V. J. & Bremer, H. Guanosine tetraphosphate (ppGpp) dependence of the growth rate control of rrnB P1 promoter activity in Escherichia coli. J. Biol. Chem. 265, 11605–11614 (1990).

    CAS  PubMed  Google Scholar 

  41. 41.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Shibuya, M. & Kaziro, Y. Studies on stringent control in a cell-free system. Regulation by guanosine-5′-diphosphate-3′-diphosphate of the synthesis of elongation factor Tu. J. Biochem. 86, 403–411 (1979).

    CAS  PubMed  Google Scholar 

  43. 43.

    Zhu, M. & Dai, X. Growth suppression by altered (p)ppGpp levels results from non-optimal resource allocation in Escherichia coli. Nucleic Acids Res. 47, 4684–4693 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Scott, M., Klumpp, S., Mateescu, E. M. & Hwa, T. Emergence of robust growth laws from optimal regulation of ribosome synthesis. Mol. Syst. Biol. 10, 747 (2014).

    PubMed  PubMed Central  Google Scholar 

  45. 45.

    Siibak, T. et al. Erythromycin- and chloramphenicol-induced ribosomal assembly defects are secondary effects of protein synthesis inhibition. Antimicrob. Agents Chemother. 53, 563–571 (2009).

    CAS  PubMed  Google Scholar 

  46. 46.

    Scott, M., Gunderson, C. W., Mateescu, E. M., Zhang, Z. & Hwa, T. Interdependence of cell growth and gene expression: origins and consequences. Science 330, 1099–1102 (2010).

    CAS  PubMed  Google Scholar 

  47. 47.

    Saxena, S. & Gowrishankar, J. Modulation of Rho-dependent transcription termination in Escherichia coli by the H-NS family of proteins. J. Bacteriol. 193, 3832–3841 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Nieto, J. M., Bailey, M. J., Hughes, C. & Koronakis, V. Suppression of transcription polarity in the Escherichia coli haemolysin operon by a short upstream element shared by polysaccharide and DNA transfer determinants. Mol. Microbiol. 19, 705–713 (1996).

    CAS  PubMed  Google Scholar 

  49. 49.

    Cayley, S., Lewis, B. A., Guttman, H. J. & Record, M. T. Jr. Characterization of the cytoplasm of Escherichia coli K-12 as a function of external osmolarity. Implications for protein-DNA interactions in vivo. J. Mol. Biol. 222, 281–300 (1991).

    CAS  PubMed  Google Scholar 

  50. 50.

    Ihara, Y., Ohta, H. & Masuda, S. A highly sensitive quantification method for the accumulation of alarmone ppGpp in Arabidopsis thaliana using UPLC-ESI-qMS/MS. J. Plant Res. 128, 511–518 (2015).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to M. Kim and R. Balakrishnan for discussions. X.D. and M.Z. acknowledge the support of the National Natural Science Fund of China (grant Nos. 31700089, 31700039 and 31870028) and by CCNU (self-determined research funds of CCNU from the college’s basic research and operation of MOE, grant Nos. CCNU18QN028, CCNU18KFY01, CCNU19TS028 and CCNU18ZDPY05). M.M. and T.H. acknowledge the support of the NIH through grant No. R01GM095903.

Author information

Affiliations

Authors

Contributions

X.D. and T.H. designed and supervised the study. M.Z. and X.D. performed all the experiments including strain construction, cell growth, RT–qPCR-based transcriptional kinetics, translational kinetics and RNA and total protein quantification. M.Z., X.D. and T.H. analysed the experimental data. X.D., T.H. and M.Z. wrote the main text and Supplementary information. M.M. analysed the data of premature transcriptional termination and wrote the Supplementary notes.

Corresponding authors

Correspondence to Terence Hwa or Xiongfeng Dai.

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 Notes, Supplementary Tables 1 and 2, Supplementary Figs. 1–19 and Supplementary References.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhu, M., Mori, M., Hwa, T. et al. Disruption of transcription–translation coordination in Escherichia coli leads to premature transcriptional termination. Nat Microbiol 4, 2347–2356 (2019). https://doi.org/10.1038/s41564-019-0543-1

Download citation

Further reading