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.
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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).
Watanabe, K. et al. Total biosynthesis of antitumor nonribosomal peptides in Escherichia coli. Nat. Chem. Biol. 2, 423–428 (2006).
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).
Peters, J.M., Vangeloff, A.D. & Landick, R. Bacterial transcription terminators: the RNA 3′-end chronicles. J. Mol. Biol. 412, 793–813 (2011).
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).
Epshtein, V., Cardinale, C.J., Ruckenstein, A.E., Borukhov, S. & Nudler, E. An allosteric path to transcription termination. Mol. Cell 28, 991–1001 (2007).
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).
Sugimoto, N. et al. Thermodynamic parameters to predict stability. Biochemistry 34, 11211–11216 (1995).
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).
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).
Gusarov, I. & Nudler, E. The mechanism of intrinsic transcription termination. Mol. Cell 3, 495–504 (1999).
Macdonald, L.E., Zhou, Y. & McAllister, W.T. Termination and slippage by bacteriophage T7 RNA polymerase. J. Mol. Biol. 232, 1030–1047 (1993).
Argaman, L. et al. Novel small RNA-encoding genes in the intergenic regions of Escherichia coli. Curr. Biol. 11, 941–950 (2001).
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).
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).
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).
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).
Lesnik, E.A. et al. Prediction of rho-independent transcriptional terminators in Escherichia coli. Nucleic Acids Res. 29, 3583–3594 (2001).
Mitra, A., Angamuthu, K., Jayashree, H.V. & Nagaraja, V. Occurrence, divergence and evolution of intrinsic terminators across eubacteria. Genomics 94, 110–116 (2009).
Mitra, A., Kesarwani, A.K., Pal, D. & Nagaraja, V. WebGeSTer DB—a transcription terminator database. Nucleic Acids Res. 39, D129–D135 (2011).
Unniraman, S., Prakash, R. & Nagaraja, V. Conserved economics of transcription termination in eubacteria. Nucleic Acids Res. 30, 675–684 (2002).
Yager, T.D. & von Hippel, P.H. A thermodynamic analysis of RNA transcript elongation and termination in Escherichia coli. Biochemistry 30, 1097–1118 (1991).
von Hippel, P.H. & Yager, T.D. Transcript elongation and termination are competitive kinetic processes. Proc. Natl. Acad. Sci. USA 88, 2307–2311 (1991).
Cambray, G. et al. Measurement and modeling of intrinsic transcription terminators. Nucleic Acids Res. 41, 5139–5148 (2013).
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).
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).
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).
Huang, H. Design and Characterization of Artificial Transcriptional Terminators. Master's thesis, Massachusetts Institute of Technology (2007).
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).
Hofacker, I.L. et al. Fast folding and comparison of RNA secondary structures. Monatshefte Chemie 125, 167–188 (1994).
Varani, G. Exceptionally stable nucleic acid hairpins. Annu. Rev. Biophys. Biomol. Struct. 24, 379–404 (1995).
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).
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).
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).
Tholstrup, J., Oddershede, L.B. & Sørensen, M.A. mRNA pseudoknot structures can act as ribosomal roadblocks. Nucleic Acids Res. 40, 303–313 (2012).
Siuti, P., Yazbek, J. & Lu, T.K. Synthetic circuits integrating logic and memory in living cells. Nat. Biotechnol. 31, 448–452 (2013).
Fujitani, Y., Yamamoto, K. & Kobayashi, I. Dependence of frequency of homologous recombination on the homology length. Genetics 140, 797–809 (1995).
Shen, P. & Huang, H.V. Homologous recombination in Escherichia coli: dependence on substrate length and homology. Genetics 112, 441–457 (1986).
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).
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).
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).
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.
K.C. and T.P. are employees of Life Technologies, which funded this work and commercializes the terminator libraries and assays described.
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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
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