De novo design of heat-repressible RNA thermosensors in E. coli

RNA-based temperature sensing is common in bacteria that live in fluctuating environments. Most naturally-occurring RNA thermosensors are heat-inducible, have long sequences, and function by sequestering the ribosome binding site in a hairpin structure at lower temperatures. Here, we demonstrate the de novo design of short, heat-repressible RNA thermosensors. These thermosensors contain a cleavage site for RNase E, an enzyme native to Escherichia coli and many other organisms, in the 5′ untranslated region of the target gene. At low temperatures, the cleavage site is sequestered in a stem–loop, and gene expression is unobstructed. At high temperatures, the stem–loop unfolds, allowing for mRNA degradation and turning off expression. We demonstrated that these thermosensors respond specifically to temperature and provided experimental support for the central role of RNase E in the mechanism. We also demonstrated the modularity of these RNA thermosensors by constructing a three-input composite circuit that utilizes transcriptional, post-transcriptional, and post-translational regulation. A thorough analysis of the 24 thermosensors allowed for the development of design guidelines for systematic construction of similar thermosensors in future applications. These short, modular RNA thermosensors can be applied to the construction of complex genetic circuits, facilitating rational reprogramming of cellular processes for synthetic biology applications.

[1]  Yiqing Shen,et al.  Configurational diffusion down a folding funnel describes the dynamics of DNA hairpins , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[2]  G. Hong,et al.  Nucleic Acids Research , 2015, Nucleic Acids Research.

[3]  Kyle E. Watters,et al.  The centrality of RNA for engineering gene expression , 2013, Biotechnology journal.

[4]  S. Gygi,et al.  Correlation between Protein and mRNA Abundance in Yeast , 1999, Molecular and Cellular Biology.

[5]  M. Inouye,et al.  The cold‐shock response — a hot topic , 1994, Molecular microbiology.

[6]  J. Russell,et al.  The ability of Escherichia coli O157:H7 to decrease its intracellular pH and resist the toxicity of acetic acid. , 1997, Microbiology.

[7]  A. J. Carpousis,et al.  A tale of two mRNA degradation pathways mediated by RNase E , 2011, Molecular microbiology.

[8]  Christopher A. Voigt,et al.  Induction and relaxation dynamics of the regulatory network controlling the type III secretion system encoded within Salmonella pathogenicity island 1. , 2008, Journal of molecular biology.

[9]  Shizhong Li,et al.  Numerical simulation of transient radial temperature distribution in rotating drum bioreactor for solid state fermentation , 2013, 2013 International Conference on Materials for Renewable Energy and Environment.

[10]  C. Gualerzi,et al.  The cspA mRNA is a thermosensor that modulates translation of the cold-shock protein CspA. , 2010, Molecular cell.

[11]  Brian F. Pfleger,et al.  Genetic and genomic analysis of RNases in model cyanobacteria , 2015, Photosynthesis Research.

[12]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[13]  Schuyler F. Baldwin,et al.  The Complete Genome Sequence of Escherichia coli DH10B: Insights into the Biology of a Laboratory Workhorse , 2008, Journal of bacteriology.

[14]  Richard M. Murray,et al.  Rapidly Characterizing the Fast Dynamics of RNA Genetic Circuitry with Cell-Free Transcription–Translation (TX-TL) Systems , 2014, ACS synthetic biology.

[15]  Carola Engler,et al.  A One Pot, One Step, Precision Cloning Method with High Throughput Capability , 2008, PloS one.

[16]  Christopher A. Voigt,et al.  Genetic programs constructed from layered logic gates in single cells , 2012, Nature.

[17]  Christopher A. Voigt,et al.  Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks , 2014, Molecular systems biology.

[18]  F. Narberhaus,et al.  Molecular basis for temperature sensing by an RNA thermometer , 2006, The EMBO journal.

[19]  É. Massé,et al.  Dual-acting riboswitch control of translation initiation and mRNA decay , 2012, Proceedings of the National Academy of Sciences.

[20]  S. Gottesman,et al.  Signal Transduction Cascade for Regulation of RpoS: Temperature Regulation of DsrA , 2001, Journal of bacteriology.

[21]  Adam P. Arkin,et al.  The Case for RNA , 2010, Science.

[22]  Erik Willems,et al.  Standardization of real-time PCR gene expression data from independent biological replicates. , 2008, Analytical biochemistry.

[23]  F. Narberhaus,et al.  Bacterial RNA thermometers: molecular zippers and switches , 2012, Nature Reviews Microbiology.

[24]  H. Hennecke,et al.  A mRNA-based thermosensor controls expression of rhizobial heat shock genes. , 2001, Nucleic acids research.

[25]  P. Højrup,et al.  Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction. , 2002, Molecular cell.

[26]  M. Serra,et al.  Stability of RNA hairpins closed by wobble base pairs. , 1998, Biochemistry.

[27]  John W. Foster,et al.  Escherichia coli acid resistance: tales of an amateur acidophile , 2004, Nature Reviews Microbiology.

[28]  Elizabeth A. Craig,et al.  Heat shock proteins and molecular chaperones: Mediators of protein conformation and turnover in the cell , 1994, Cell.

[29]  Christopher A. Voigt,et al.  Automated design of synthetic ribosome binding sites to control protein expression , 2016 .

[30]  Juliane Neupert,et al.  Design of simple synthetic RNA thermometers for temperature-controlled gene expression in Escherichia coli , 2008, Nucleic acids research.

[31]  D. Crothers,et al.  Conformational changes of transfer ribonucleic acid. Equilibrium phase diagrams. , 1972, Biochemistry.

[32]  David E Draper,et al.  A guide to ions and RNA structure. , 2004, RNA.

[33]  Elisa Franco,et al.  Dynamically Reshaping Signaling Networks to Program Cell Fate via Genetic Controllers , 2013, Science.

[34]  G. Horn,et al.  Structure and function of bacterial cold shock proteins , 2007, Cellular and Molecular Life Sciences.

[35]  F. Narberhaus,et al.  Thermozymes: Synthetic RNA thermometers based on ribozyme activity. , 2013, RNA biology.

[36]  Dmitrij Frishman,et al.  RNAtips: analysis of temperature-induced changes of RNA secondary structure , 2013, Nucleic Acids Res..

[37]  S. Woodson,et al.  Loop dependence of the stability and dynamics of nucleic acid hairpins , 2007, Nucleic acids research.

[38]  Enhanced in vitro translation at reduced temperatures using a cold-shock RNA motif , 2013, Biotechnology Letters.

[39]  A. Muga,et al.  Thermal adaptation of heat shock proteins. , 2008, Current protein & peptide science.

[40]  Torsten Waldminghaus,et al.  RNA thermometers. , 2006, FEMS microbiology reviews.

[41]  H. Salis,et al.  Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites , 2013, Nucleic acids research.

[42]  M. Bina-Stein,et al.  Conformational changes of transfer ribonucleic acid. The pH phase diagram under acidic conditions. , 1974, Biochemistry.

[43]  Takashi Yura,et al.  Convergence of Molecular, Modeling, and Systems Approaches for an Understanding of the Escherichia coli Heat Shock Response , 2008, Microbiology and Molecular Biology Reviews.

[44]  Andrew D. Ellington,et al.  Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers , 2014, Nucleic acids research.

[45]  K. Darwin,et al.  Type III secretion chaperone‐dependent regulation: activation of virulence genes by SicA and InvF in Salmonella typhimurium , 2001, The EMBO journal.

[46]  G. Stephanopoulos,et al.  Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR , 2011, BMC Molecular Biology.

[47]  Koji Hayashi,et al.  Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110 , 2006, Molecular systems biology.

[48]  John W. Foster,et al.  Escherichia coli Glutamate- and Arginine-Dependent Acid Resistance Systems Increase Internal pH and Reverse Transmembrane Potential , 2004, Journal of bacteriology.

[49]  Shi-jie Chen,et al.  Exploring the complex folding kinetics of RNA hairpins: I. General folding kinetics analysis. , 2006, Biophysical journal.

[50]  M. Dreyfus,et al.  The C‐terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo , 1999, Molecular microbiology.

[51]  Harald Schwalbe,et al.  Modulation of the stability of the Salmonella fourU-type RNA thermometer , 2011, Nucleic acids research.

[52]  H. Mori,et al.  Regulation of the heat-shock response in bacteria. , 1993, Annual review of microbiology.

[53]  H. Lönnberg,et al.  Kinetics and Mechanisms for the Cleavage and Isomerization of the Phosphodiester Bonds of RNA by Brønsted Acids and Bases. , 1998, Chemical reviews.

[54]  H. Salis.  The ribosome binding site calculator. , 2011, Methods in enzymology.

[55]  O. Uhlenbeck,et al.  Characterization of RNA hairpin loop stability. , 1988, Nucleic acids research.

[56]  J. Collins,et al.  Toehold Switches: De-Novo-Designed Regulators of Gene Expression , 2014, Cell.

[57]  Alan Van Orden,et al.  A three-state mechanism for DNA hairpin folding characterized by multiparameter fluorescence fluctuation spectroscopy. , 2006, Journal of the American Chemical Society.

[58]  Benoit Guieysse,et al.  Mechanistic modeling of broth temperature in outdoor photobioreactors. , 2010, Environmental science & technology.

[59]  C. Condon,et al.  The phylogenetic distribution of bacterial ribonucleases. , 2002, Nucleic acids research.

[60]  Adam P Arkin,et al.  Versatile RNA-sensing transcriptional regulators for engineering genetic networks , 2011, Proceedings of the National Academy of Sciences.

[61]  B. Luisi,et al.  Endonucleolytic initiation of mRNA decay in Escherichia coli. , 2009, Progress in molecular biology and translational science.

[62]  Nicholas T. Ingolia,et al.  Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling , 2009, Science.

[63]  H. Hennecke,et al.  A novel DNA element that controls bacterial heat shock gene expression , 1998, Molecular microbiology.

[64]  L. Nissim,et al.  Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. , 2014, Molecular cell.

[65]  K. Hall,et al.  Effect of loop composition on the stability and folding kinetics of RNA hairpins with large loops. , 2015, Biochemistry.

[66]  Emma Kreuger,et al.  Temperature-controlled Structural Alterations of an RNA Thermometer* , 2003, Journal of Biological Chemistry.

[67]  J. Sabina,et al.  Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. , 1999, Journal of molecular biology.

[68]  Leonidas J. Guibas,et al.  Structural Insight into RNA Hairpin Folding Intermediates , 2008, Journal of the American Chemical Society.

[69]  V. Hatzimanikatis,et al.  Thermodynamics-based metabolic flux analysis. , 2007, Biophysical journal.

[70]  Jay D Keasling,et al.  Model-Driven Engineering of RNA Devices to Quantitatively Program Gene Expression , 2011, Science.

[71]  D. Na,et al.  Design and use of synthetic regulatory small RNAs to control gene expression in Escherichia coli , 2013, Nature Protocols.

[72]  Z. Cai,et al.  Synthesizing AND gate genetic circuits based on CRISPR-Cas9 for identification of bladder cancer cells , 2014, Nature Communications.

[73]  A. Grossman,et al.  The htpR gene product of E. coli is a sigma factor for heat-shock promoters , 1984, Cell.

[74]  Gene-Wei Li,et al.  The anti-Shine-Dalgarno sequence drives translational pausing and codon choice in bacteria , 2012, Nature.

[75]  E. Marcotte,et al.  Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation , 2007, Nature Biotechnology.

[76]  Weston R. Whitaker,et al.  Toward scalable parts families for predictable design of biological circuits. , 2008, Current opinion in microbiology.

[77]  Adam P. Arkin,et al.  A versatile framework for microbial engineering using synthetic non-coding RNAs , 2014, Nature Reviews Microbiology.

[78]  V. Beneš,et al.  The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. , 2009, Clinical chemistry.

[79]  Torsten Waldminghaus,et al.  Generation of synthetic RNA-based thermosensors , 2008, Biological chemistry.