Characterization of 582 Natural and Synthetic Terminators and Quantification of Their Design Constraints

Overview

Journal Nat Methods

Specialties Biomedical Engineering
Pathology

Date 2013 Jun 4

PMID 23727987

Citations 236

Authors

Ying-Ja Chen

Peng Liu

Alec A K Nielsen

Jennifer A N Brophy

Kevin Clancy

Todd Peterson

Christopher A Voigt

Affiliations

Soon will be listed here.

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.

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References

Epshtein V, Cardinale C, Ruckenstein A, Borukhov S, Nudler E . An allosteric path to transcription termination. Mol Cell. 2007; 28(6):991-1001. DOI: 10.1016/j.molcel.2007.10.011. View

Martin F, Tinoco Jr I . DNA-RNA hybrid duplexes containing oligo(dA:rU) sequences are exceptionally unstable and may facilitate termination of transcription. Nucleic Acids Res. 1980; 8(10):2295-9. PMC: 324078. DOI: 10.1093/nar/8.10.2295. View

Sleight S, Bartley B, Lieviant J, Sauro H . Designing and engineering evolutionary robust genetic circuits. J Biol Eng. 2010; 4:12. PMC: 2991278. DOI: 10.1186/1754-1611-4-12. View

Huang Y, Weng X, Russu I . Structural energetics of the adenine tract from an intrinsic transcription terminator. J Mol Biol. 2010; 397(3):677-88. PMC: 2908014. DOI: 10.1016/j.jmb.2010.01.068. View

Kingsford C, Ayanbule K, Salzberg S . Rapid, accurate, computational discovery of Rho-independent transcription terminators illuminates their relationship to DNA uptake. Genome Biol. 2007; 8(2):R22. PMC: 1852404. DOI: 10.1186/gb-2007-8-2-r22. View

Gusarov I, Nudler E . The mechanism of intrinsic transcription termination. Mol Cell. 1999; 3(4):495-504. DOI: 10.1016/s1097-2765(00)80477-3. View

Xayaphoummine A, Bucher T, Isambert H . Kinefold web server for RNA/DNA folding path and structure prediction including pseudoknots and knots. Nucleic Acids Res. 2005; 33(Web Server issue):W605-10. PMC: 1160208. DOI: 10.1093/nar/gki447. View

MacDonald L, Zhou Y, McAllister W . Termination and slippage by bacteriophage T7 RNA polymerase. J Mol Biol. 1993; 232(4):1030-47. DOI: 10.1006/jmbi.1993.1458. View

Lesnik E, Sampath R, Levene H, Henderson T, McNeil J, Ecker D . Prediction of rho-independent transcriptional terminators in Escherichia coli. Nucleic Acids Res. 2001; 29(17):3583-94. PMC: 55870. DOI: 10.1093/nar/29.17.3583. View

10.

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 U S A. 1978; 75(9):4271-5. PMC: 336095. DOI: 10.1073/pnas.75.9.4271. View

11.

Larson M, Greenleaf W, Landick R, Block S . Applied force reveals mechanistic and energetic details of transcription termination. Cell. 2008; 132(6):971-82. PMC: 2295211. DOI: 10.1016/j.cell.2008.01.027. View

12.

dAubenton 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. 1990; 216(4):835-58. DOI: 10.1016/s0022-2836(99)80005-9. View

13.

Kwon Y, Kang C . Bipartite modular structure of intrinsic, RNA hairpin-independent termination signal for phage RNA polymerases. J Biol Chem. 1999; 274(41):29149-55. DOI: 10.1074/jbc.274.41.29149. View

14.

Unniraman S, Prakash R, Nagaraja V . Conserved economics of transcription termination in eubacteria. Nucleic Acids Res. 2002; 30(3):675-84. PMC: 100295. DOI: 10.1093/nar/30.3.675. View

15.

Varani G . Exceptionally stable nucleic acid hairpins. Annu Rev Biophys Biomol Struct. 1995; 24:379-404. DOI: 10.1146/annurev.bb.24.060195.002115. View

16.

von Hippel P, Yager T . Transcript elongation and termination are competitive kinetic processes. Proc Natl Acad Sci U S A. 1991; 88(6):2307-11. PMC: 51220. DOI: 10.1073/pnas.88.6.2307. View

17.

Siuti P, Yazbek J, Lu T . Synthetic circuits integrating logic and memory in living cells. Nat Biotechnol. 2013; 31(5):448-52. DOI: 10.1038/nbt.2510. View

18.

Moon T, Lou C, Tamsir A, Stanton B, Voigt C . Genetic programs constructed from layered logic gates in single cells. Nature. 2012; 491(7423):249-53. PMC: 3904217. DOI: 10.1038/nature11516. View

19.

Lou C, Stanton B, Chen Y, Munsky B, Voigt C . Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nat Biotechnol. 2012; 30(11):1137-42. PMC: 3914141. DOI: 10.1038/nbt.2401. View

20.

Argaman L, HERSHBERG R, Vogel J, Bejerano G, WAGNER E, Margalit H . Novel small RNA-encoding genes in the intergenic regions of Escherichia coli. Curr Biol. 2001; 11(12):941-50. DOI: 10.1016/s0960-9822(01)00270-6. View