We expanded the mechanistic capability of small RNAs by creating an entirely synthetic mode of regulation: small transcription activating RNAs (STARs). Using two strategies, we engineered synthetic STAR regulators to disrupt the formation of an intrinsic transcription terminator placed upstream of a gene in Escherichia coli. This resulted in a group of four highly orthogonal STARs that had up to 94-fold activation. By systematically modifying sequence features of this group, we derived design principles for STAR function, which we then used to forward engineer a STAR that targets a terminator found in the Escherichia coli genome. Finally, we showed that STARs could be combined in tandem to create previously unattainable RNA-only transcriptional logic gates. STARs provide a new mechanism of regulation that will expand our ability to use small RNAs to construct synthetic gene networks that precisely control gene expression.
This is a preview of subscription content
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Chappell, J. et al. The centrality of RNA for engineering gene expression. Biotechnol. J. 8, 1379–1395 (2013).
Carothers, J.M., Goler, J.A., Juminaga, D. & Keasling, J.D. Model-driven engineering of RNA devices to quantitatively program gene expression. Science 334, 1716–1719 (2011).
Rodrigo, G., Landrain, T.E. & Jaramillo, A. De novo automated design of small RNA circuits for engineering synthetic riboregulation in living cells. Proc. Natl. Acad. Sci. USA 109, 15271–15276 (2012).
Wachsmuth, M., Findeiss, S., Weissheimer, N., Stadler, P.F. & Morl, M. De novo design of a synthetic riboswitch that regulates transcription termination. Nucleic Acids Res. 41, 2541–2551 (2013).
Xayaphoummine, A., Viasnoff, V., Harlepp, S. & Isambert, H. Encoding folding paths of RNA switches. Nucleic Acids Res. 35, 614–622 (2007).
Lucks, J.B. et al. Multiplexed RNA structure characterization with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). Proc. Natl. Acad. Sci. USA 108, 11063–11068 (2011).
Rouskin, S., Zubradt, M., Washietl, S., Kellis, M. & Weissman, J.S. Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo. Nature 505, 701–705 (2014).
Storz, G., Vogel, J. & Wassarman, K.M. Regulation by small RNAs in bacteria: expanding frontiers. Mol. Cell 43, 880–891 (2011).
Na, D. et al. Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nat. Biotechnol. 31, 170–174 (2013).
Sharma, V., Yamamura, A. & Yokobayashi, Y. Engineering artificial small RNAs for conditional gene silencing in Escherichia coli. ACS Synth. Biol. 1, 6–13 (2012).
Callura, J.M., Cantor, C.R. & Collins, J.J. Genetic switchboard for synthetic biology applications. Proc. Natl. Acad. Sci. USA 109, 5850–5855 (2012).
Friedland, A.E. et al. Synthetic gene networks that count. Science 324, 1199–1202 (2009).
Isaacs, F.J. et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat. Biotechnol. 22, 841–847 (2004).
Lucks, J.B., Qi, L., Mutalik, V.K., Wang, D. & Arkin, A.P. Versatile RNA-sensing transcriptional regulators for engineering genetic networks. Proc. Natl. Acad. Sci. USA 108, 8617–8622 (2011).
Takahashi, M.K. & Lucks, J.B. A modular strategy for engineering orthogonal chimeric RNA transcription regulators. Nucleic Acids Res. 41, 7577–7588 (2013).
Takahashi, M.K. et al. Rapidly characterizing the fast dynamics of RNA genetic circuitry with cell-free transcription-translation (TX-TL) systems. ACS Synth. Biol. doi:10.1021/sb400206c (12 March 2014).
Brantl, S. Regulatory mechanisms employed by cis-encoded antisense RNAs. Curr. Opin. Microbiol. 10, 102–109 (2007).
Brantl, S. & Wagner, E.G. Antisense RNA-mediated transcriptional attenuation: an in vitro study of plasmid pT181. Mol. Microbiol. 35, 1469–1482 (2000).
Kumar, C.C. & Novick, R.P. Plasmid pT181 replication is regulated by two countertranscripts. Proc. Natl. Acad. Sci. USA 82, 638–642 (1985).
Qi, L., Lucks, J.B., Liu, C.C., Mutalik, V.K. & Arkin, A.P. Engineering naturally occurring trans-acting non-coding RNAs to sense molecular signals. Nucleic Acids Res. 40, 5775–5786 (2012).
Sakai, Y. et al. Improving the gene-regulation ability of small RNAs by scaffold engineering in Escherichia coli. ACS Synth. Biol. 3, 152–162 (2014).
Dawid, A., Cayrol, B. & Isambert, H. RNA synthetic biology inspired from bacteria: construction of transcription attenuators under antisense regulation. Phys. Biol. 6, 025007 (2009).
Pédelacq, J.D., Cabantous, S., Tran, T., Terwilliger, T.C. & Waldo, G.S. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88 (2006).
Ceres, P., Trausch, J.J. & Batey, R.T. Engineering modular 'ON' RNA switches using biological components. Nucleic Acids Res. 41, 10449–10461 (2013).
Ceres, P., Garst, A.D., Marcano-Velazquez, J.G. & Batey, R.T. Modularity of select riboswitch expression platforms enables facile engineering of novel genetic regulatory devices. ACS Synth. Biol. 2, 463–472 (2013).
Sun, Z.Z. et al. Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology. J. Vis. Exp. 2013, 16 e50762 (2013).
Shin, J. & Noireaux, V. An E. coli cell-free expression toolbox: application to synthetic gene circuits and artificial cells. ACS Synth. Biol. 1, 29–41 (2012).
Sun, Z.Z., Yeung, E., Hayes, C.A., Noireaux, V. & Murray, R.M. Linear DNA for rapid prototyping of synthetic biological circuits in an Escherichia coli based TX-TL cell-free system. ACS Synth. Biol. 3, 387–397 (2014).
Chappell, J., Jensen, K. & Freemont, P.S. Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology. Nucleic Acids Res. 41, 3471–3481 (2013).
Salis, H.M., Mirsky, E.A. & Voigt, C.A. Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27, 946–950 (2009).
Mutalik, V.K., Qi, L., Guimaraes, J.C., Lucks, J.B. & Arkin, A.P. Rationally designed families of orthogonal RNA regulators of translation. Nat. Chem. Biol. 8, 447–454 (2012).
Green, A.A., Silver, P.A., Collins, J.J. & Yin, P. Toehold switches: de-novo-designed regulators of gene expression. Cell 159, 925–939 (2014).
Kolb, F.A. et al. Progression of a loop-loop complex to a four-way junction is crucial for the activity of a regulatory antisense RNA. EMBO J. 19, 5905–5915 (2000).
Anderson, J.C., Voigt, C.A. & Arkin, A.P. Environmental signal integration by a modular AND gate. Mol. Syst. Biol. 3, 133 (2007).
Ellis, T., Wang, X. & Collins, J.J. Diversity-based, model-guided construction of synthetic gene networks with predicted functions. Nat. Biotechnol. 27, 465–471 (2009).
Kotula, J.W. et al. Programmable bacteria detect and record an environmental signal in the mammalian gut. Proc. Natl. Acad. Sci. USA 111, 4838–4843 (2014).
Liu, C.C. et al. An adaptor from translational to transcriptional control enables predictable assembly of complex regulation. Nat. Methods 9, 1088–1094 (2012).
Qi, L.S. & Arkin, A.P. A versatile framework for microbial engineering using synthetic non-coding RNAs. Nat. Rev. Microbiol. 12, 341–354 (2014).
Mathews, D.H. & Turner, D.H. Prediction of RNA secondary structure by free energy minimization. Curr. Opin. Struct. Biol. 16, 270–278 (2006).
Bikard, D. et al. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 41, 7429–7437 (2013).
Qi, L.S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).
Farzadfard, F., Perli, S.D. & Lu, T.K. Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. ACS Synth. Biol. 2, 604–613 (2013).
Nissim, L., Perli, S.D., Fridkin, A., Perez-Pinera, P. & Lu, T.K. Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol. Cell 54, 698–710 (2014).
Kiani, S. et al. CRISPR transcriptional repression devices and layered circuits in mammalian cells. Nat. Methods 11, 723–726 (2014).
Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).
Goff, L., Trapnell, C. & Kelley, D. cummeRbund: analysis, exploration, manipulation, and visualization of Cufflinks high-throughput sequencing data. (R package version 2.6.1, 2012).
Bellaousov, S., Reuter, J.S., Seetin, M.G. & Mathews, D.H. RNAstructure: Web servers for RNA secondary structure prediction and analysis. Nucleic Acids Res. 41, W471–W474 (2013).
The authors acknowledge J. Roberts, E. Strobel, A. Stroock and the Lucks Lab members for helpful discussions. We would also like to thank C. Trapnell for help with RNA-seq experimental design and analysis. We also thank J. Peters (Department of Microbiology, Cornell University) for providing E. coli strain K12 MG1655. Finally, we would like to thank D. Tapias-Rojas for preliminary work on targeting naturally occurring intrinsic terminators. This material is based on work supported by the National Science Foundation Graduate Research Fellowship Program (grant no. DGE-1144153 to M.K.T.), the Defense Advanced Research Projects Agency Young Faculty Award (DARPA YFA; no. N66001-12-1-4254 to J.B.L.) and an Office of Naval Research Young Investigators Program Award (ONR YIP; no. N00014-13-1-0531 to J.B. L.). J.B.L. is an Alfred P. Sloan Research Fellow.
The authors have submitted a provisional patent application (No. 61/981,241) for the technologically important developments included in this Article.
About this article
Cite this article
Chappell, J., Takahashi, M. & Lucks, J. Creating small transcription activating RNAs. Nat Chem Biol 11, 214–220 (2015). https://doi.org/10.1038/nchembio.1737
Multi-arm RNA junctions encoding molecular logic unconstrained by input sequence for versatile cell-free diagnostics
Nature Biomedical Engineering (2022)
Applied Microbiology and Biotechnology (2022)
Nature Communications (2021)
Nature Chemical Biology (2021)