Characterization of 582 natural and synthetic terminators and quantification of their design constraints

Abstract

Large genetic engineering projects require more cistrons and consequently more strong and reliable transcriptional terminators. We have measured the strengths of a library of terminators, including 227 that are annotated in Escherichia coli—90 of which we also tested in the reverse orientation—and 265 synthetic terminators. Within this library we found 39 strong terminators, yielding >50-fold reduction in downstream expression, that have sufficient sequence diversity to reduce homologous recombination when used together in a design. We used these data to determine how the terminator sequence contributes to its strength. The dominant parameters were incorporated into a biophysical model that considers the role of the hairpin in the displacement of the U-tract from the DNA. The availability of many terminators of varying strength, as well as an understanding of the sequence dependence of their properties, will extend their usability in the forward design of synthetic cistrons.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Measurement of terminator strength for the natural and synthetic libraries.
Figure 2: Sequence features that contribute to terminator strength.
Figure 3: Impact of changing the various components of a terminator in three strong terminator 'scaffolds'.
Figure 4: Biophysical model of terminator strength, TS, and recombination propensity.

References

  1. 1

    Moon, T.S., Lou, C., Tamsir, A., Stanton, B.C. & Voigt, C.A. Genetic programs constructed from layered logic gates in single cells. Nature 491, 249–253 (2012).

    CAS  Article  Google Scholar 

  2. 2

    Watanabe, K. et al. Total biosynthesis of antitumor nonribosomal peptides in Escherichia coli. Nat. Chem. Biol. 2, 423–428 (2006).

    CAS  Article  Google Scholar 

  3. 3

    Sleight, S.C., Bartley, B.A., Lieviant, J.A. & Sauro, H.M. Designing and engineering evolutionary robust genetic circuits. J. Biol. Eng. 4, 12 (2010).

    Article  Google Scholar 

  4. 4

    Peters, J.M., Vangeloff, A.D. & Landick, R. Bacterial transcription terminators: the RNA 3′-end chronicles. J. Mol. Biol. 412, 793–813 (2011).

    CAS  Article  Google Scholar 

  5. 5

    Komissarova, N., Becker, J., Solter, S., Kireeva, M. & Kashlev, M. Shortening of RNA:DNA hybrid in the elongation complex of RNA polymerase is a prerequisite for transcription termination. Mol. Cell 10, 1151–1162 (2002).

    CAS  Article  Google Scholar 

  6. 6

    Epshtein, V., Cardinale, C.J., Ruckenstein, A.E., Borukhov, S. & Nudler, E. An allosteric path to transcription termination. Mol. Cell 28, 991–1001 (2007).

    CAS  Article  Google Scholar 

  7. 7

    Martin, F.H. & Tinoco, I. DNA-RNA hybrid duplexes containing oligo(dA:rU) sequences are exceptionally unstable and may facilitate termination of transcription. Nucleic Acids Res. 8, 2295–2299 (1980).

    CAS  Article  Google Scholar 

  8. 8

    Sugimoto, N. et al. Thermodynamic parameters to predict stability. Biochemistry 34, 11211–11216 (1995).

    CAS  Article  Google Scholar 

  9. 9

    Huang, Y., Chen, C. & Russu, I.M. Dynamics and stability of individual base pairs in two homologous RNA-DNA hybrids. Biochemistry 48, 3988–3997 (2009).

    CAS  Article  Google Scholar 

  10. 10

    Huang, Y., Weng, X. & Russu, I.M. Structural energetics of the adenine tract from an intrinsic transcription terminator. J. Mol. Biol. 397, 677–688 (2010).

    CAS  Article  Google Scholar 

  11. 11

    Gusarov, I. & Nudler, E. The mechanism of intrinsic transcription termination. Mol. Cell 3, 495–504 (1999).

    CAS  Article  Google Scholar 

  12. 12

    Macdonald, L.E., Zhou, Y. & McAllister, W.T. Termination and slippage by bacteriophage T7 RNA polymerase. J. Mol. Biol. 232, 1030–1047 (1993).

    CAS  Article  Google Scholar 

  13. 13

    Argaman, L. et al. Novel small RNA-encoding genes in the intergenic regions of Escherichia coli. Curr. Biol. 11, 941–950 (2001).

    CAS  Article  Google Scholar 

  14. 14

    d'Aubenton Carafa, Y., Brody, E. & Thermes, C. Prediction of rho-independent Escherichia coli transcription terminators. A statistical analysis of their RNA stem-loop structures. J. Mol. Biol. 216, 835–858 (1990).

    CAS  Article  Google Scholar 

  15. 15

    de Hoon, M.J.L., Makita, Y., Nakai, K. & Miyano, S. Prediction of transcriptional terminators in Bacillus subtilis and related species. PLoS Comput. Biol. 1, e25 (2005).

    Article  Google Scholar 

  16. 16

    Gardner, P.P., Barquist, L., Bateman, A., Nawrocki, E.P. & Weinberg, Z. RNIE: genome-wide prediction of bacterial intrinsic terminators. Nucleic Acids Res. 39, 5845–5852 (2011).

    CAS  Article  Google Scholar 

  17. 17

    Kingsford, C.L., Ayanbule, K. & Salzberg, S.L. Rapid, accurate, computational discovery of rho-independent transcription terminators illuminates their relationship to DNA uptake. Genome Biol. 8, R22 (2007).

    Article  Google Scholar 

  18. 18

    Lesnik, E.A. et al. Prediction of rho-independent transcriptional terminators in Escherichia coli. Nucleic Acids Res. 29, 3583–3594 (2001).

    CAS  Article  Google Scholar 

  19. 19

    Mitra, A., Angamuthu, K., Jayashree, H.V. & Nagaraja, V. Occurrence, divergence and evolution of intrinsic terminators across eubacteria. Genomics 94, 110–116 (2009).

    CAS  Article  Google Scholar 

  20. 20

    Mitra, A., Kesarwani, A.K., Pal, D. & Nagaraja, V. WebGeSTer DB—a transcription terminator database. Nucleic Acids Res. 39, D129–D135 (2011).

    CAS  Article  Google Scholar 

  21. 21

    Unniraman, S., Prakash, R. & Nagaraja, V. Conserved economics of transcription termination in eubacteria. Nucleic Acids Res. 30, 675–684 (2002).

    CAS  Article  Google Scholar 

  22. 22

    Yager, T.D. & von Hippel, P.H. A thermodynamic analysis of RNA transcript elongation and termination in Escherichia coli. Biochemistry 30, 1097–1118 (1991).

    CAS  Article  Google Scholar 

  23. 23

    von Hippel, P.H. & Yager, T.D. Transcript elongation and termination are competitive kinetic processes. Proc. Natl. Acad. Sci. USA 88, 2307–2311 (1991).

    CAS  Article  Google Scholar 

  24. 24

    Cambray, G. et al. Measurement and modeling of intrinsic transcription terminators. Nucleic Acids Res. 41, 5139–5148 (2013).

    CAS  Article  Google Scholar 

  25. 25

    Kwon, Y.S. & Kang, C. Bipartite modular structure of intrinsic, RNA hairpin-independent termination signal for phage RNA polymerases. J. Biol. Chem. 274, 29149–29155 (1999).

    CAS  Article  Google Scholar 

  26. 26

    Gama-Castro, S. et al. RegulonDB version 7.0: transcriptional regulation of Escherichia coli K-12 integrated within genetic sensory response units (Gensor Units). Nucleic Acids Res. 39, D98–D105 (2011).

    CAS  Article  Google Scholar 

  27. 27

    Gama-Castro, S. et al. RegulonDB (version 6.0): gene regulation model of Escherichia coli K-12 beyond transcription, active (experimental) annotated promoters and Textpresso navigation. Nucleic Acids Res. 36, D120–D124 (2008).

    CAS  Article  Google Scholar 

  28. 28

    Huang, H. Design and Characterization of Artificial Transcriptional Terminators. Master's thesis, Massachusetts Institute of Technology (2007).

  29. 29

    Xayaphoummine, A., Bucher, T. & Isambert, H. Kinefold web server for RNA/DNA folding path and structure prediction including pseudoknots and knots. Nucleic Acids Res. 33, W605–W610 (2005).

    CAS  Article  Google Scholar 

  30. 30

    Hofacker, I.L. et al. Fast folding and comparison of RNA secondary structures. Monatshefte Chemie 125, 167–188 (1994).

    CAS  Article  Google Scholar 

  31. 31

    Varani, G. Exceptionally stable nucleic acid hairpins. Annu. Rev. Biophys. Biomol. Struct. 24, 379–404 (1995).

    CAS  Article  Google Scholar 

  32. 32

    Larson, M.H., Greenleaf, W.J., Landick, R. & Block, S.M. Applied force reveals mechanistic and energetic details of transcription termination. Cell 132, 971–982 (2008).

    CAS  Article  Google Scholar 

  33. 33

    Zurawski, G., Brown, K., Killingly, D. & Yanofsky, C. Nucleotide sequence of the leader region of the phenylalanine operon of Escherichia coli. Proc. Natl. Acad. Sci. USA 75, 4271–4275 (1978).

    CAS  Article  Google Scholar 

  34. 34

    Lopatovskaya, K.V., Seliverstov, A.V. & Lyubetsky, V.A. Attenuation regulation of the amino acid and aminoacyl-tRNA biosynthesis operons in bacteria: a comparative genomic analysis. Mol. Biol. 44, 128–139 (2010).

    CAS  Article  Google Scholar 

  35. 35

    Tholstrup, J., Oddershede, L.B. & Sørensen, M.A. mRNA pseudoknot structures can act as ribosomal roadblocks. Nucleic Acids Res. 40, 303–313 (2012).

    CAS  Article  Google Scholar 

  36. 36

    Siuti, P., Yazbek, J. & Lu, T.K. Synthetic circuits integrating logic and memory in living cells. Nat. Biotechnol. 31, 448–452 (2013).

    CAS  Article  Google Scholar 

  37. 37

    Fujitani, Y., Yamamoto, K. & Kobayashi, I. Dependence of frequency of homologous recombination on the homology length. Genetics 140, 797–809 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Shen, P. & Huang, H.V. Homologous recombination in Escherichia coli: dependence on substrate length and homology. Genetics 112, 441–457 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Lovett, S.T., Luisi-DeLuca, C. & Kolodner, R.D. The genetic dependence of recombination in recD mutants of Escherichia coli. Genetics 120, 37–45 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Temme, K., Hill, R., Segall-Shapiro, T.H., Moser, F. & Voigt, C.A. Modular control of multiple pathways using engineered orthogonal T7 polymerases. Nucleic Acids Res. 40, 8773–8781 (2012).

    CAS  Article  Google Scholar 

  41. 41

    Lou, C., Stanton, B., Chen, Y.-J., Munsky, B. & Voigt, C.A. Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nat. Biotechnol. 30, 1137–1142 (2012).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

C.A.V., P.L., A.A.K.N., J.A.N.B. and Y.-J.C. are supported by Life Technologies and the US National Science Foundation Synthetic Biology Engineering Research Center (SynBERC). Y.-J.C. thanks the PhRMA Foundation for a Postdoctoral Fellowship in Informatics. We thank R. Landick and D.Y. Zhang for their advanced review for the manuscript.

Author information

Affiliations

Authors

Contributions

C.A.V. conceived and supervised the project. Y.-J.C., A.A.K.N. and J.A.N.B. designed and performed the experiments. C.A.V., P.L. and Y.-J.C. constructed the biophysical model. K.C. and T.P. oversaw the project. C.A.V., Y.-J.C. and P.L. wrote the manuscript.

Corresponding author

Correspondence to Christopher A Voigt.

Ethics declarations

Competing interests

K.C. and T.P. are employees of Life Technologies, which funded this work and commercializes the terminator libraries and assays described.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–23, Supplementary Table 1 and Supplementary Notes 1–11 (PDF 1755 kb)

Supplementary Table 2

Natural terminators (XLSX 130 kb)

Supplementary Table 3

Synthetic terminators (XLSX 94 kb)

Supplementary Table 4

Removed terminators (XLSX 18 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Chen, YJ., Liu, P., Nielsen, A. et al. Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat Methods 10, 659–664 (2013). https://doi.org/10.1038/nmeth.2515

Download citation

Further reading

Search

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