Antisense transcription as a tool to tune gene expression

A surprise that has emerged from transcriptomics is the prevalence of genomic antisense transcription, which occurs counter to gene orientation. While frequent, the roles of antisense transcription in regulation are poorly understood. We built a synthetic system in Escherichia coli to study how antisense transcription can change the expression of a gene and tune the response characteristics of a regulatory circuit. We developed a new genetic part that consists of a unidirectional terminator followed by a constitutive antisense promoter and demonstrate that this part represses gene expression proportionally to the antisense promoter strength. Chip‐based oligo synthesis was applied to build a large library of 5,668 terminator–promoter combinations that was used to control the expression of three repressors (PhlF, SrpR, and TarA) in a simple genetic circuit (NOT gate). Using the library, we demonstrate that antisense promoters can be used to tune the threshold of a regulatory circuit without impacting other properties of its response function. Finally, we determined the relative contributions of antisense RNA and transcriptional interference to repressing gene expression and introduce a biophysical model to capture the impact of RNA polymerase collisions on gene repression. This work quantifies the role of antisense transcription in regulatory networks and introduces a new mode to control gene expression that has been previously overlooked in genetic engineering.

[1]  M. Ehrenberg,et al.  Activities of constitutive promoters in Escherichia coli. , 1999, Journal of molecular biology.

[2]  A. Arkin,et al.  Sequestration-based bistability enables tuning of the switching boundaries and design of a latch , 2012, Molecular systems biology.

[3]  J. Gowrishankar,et al.  R-loops in bacterial transcription , 2013, Transcription.

[4]  Drew Endy,et al.  Precise and reliable gene expression via standard transcription and translation initiation elements , 2013, Nature Methods.

[5]  G. Storz,et al.  An antisense RNA controls synthesis of an SOS-induced toxin evolved from an antitoxin , 2007, Molecular microbiology.

[6]  F. Bontems,et al.  Expression of highly toxic genes in E. coli: special strategies and genetic tools. , 2006, Current protein & peptide science.

[7]  T. Link,et al.  Hfq structure, function and ligand binding. , 2007, Current opinion in microbiology.

[8]  B. Connolly,et al.  The chemical synthesis of a gene coding for bovine pancreatic DNase I and its cloning and expression in Escherichia coli. , 1990, The Journal of biological chemistry.

[9]  J. Helmann,et al.  Extracytoplasmic Function σ Factors Regulate Expression of the Bacillus subtilis yabE Gene via a cis-Acting Antisense RNA , 2008, Journal of bacteriology.

[10]  M. Suyama,et al.  Transcriptome Complexity in a Genome-Reduced Bacterium , 2009, Science.

[11]  M. Elowitz,et al.  Programming gene expression with combinatorial promoters , 2007, Molecular systems biology.

[12]  A. Helmrich,et al.  Transcription-replication encounters, consequences and genomic instability , 2013, Nature Structural &Molecular Biology.

[13]  K. Drlica,et al.  DNA supercoiling and prokaryotic transcription , 1989, Cell.

[14]  G. Storz,et al.  Small Toxic Proteins and the Antisense RNAs That Repress Them , 2008, Microbiology and Molecular Biology Reviews.

[15]  J. Keasling,et al.  Engineering mRNA stability in E. coli by the addition of synthetic hairpins using a 5' cassette system. , 1997, Biotechnology and bioengineering.

[16]  P. Burguière,et al.  S-box and T-box riboswitches and antisense RNA control a sulfur metabolic operon of Clostridium acetobutylicum , 2008, Nucleic acids research.

[17]  Christopher A. Voigt,et al.  Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters , 2013, Molecular Systems Biology.

[18]  D. Lindell,et al.  Antisense RNA protects mRNA from RNase E degradation by RNA–RNA duplex formation during phage infection , 2011, Nucleic acids research.

[19]  David Hernández,et al.  Cartography of Methicillin-Resistant S. aureus Transcripts: Detection, Orientation and Temporal Expression during Growth Phase and Stress Conditions , 2010, PloS one.

[20]  Wei-Shou Hu,et al.  Convergent transcription confers a bistable switch in Enterococcus faecalis conjugation , 2011, Proceedings of the National Academy of Sciences.

[21]  Robert G. Martin,et al.  Detection of low-level promoter activity within open reading frame sequences of Escherichia coli , 2005, Nucleic acids research.

[22]  A. Brandi,et al.  A novel antisense RNA regulates at transcriptional level the virulence gene icsA of Shigella flexneri , 2010, Nucleic acids research.

[23]  S. Brantl.  Regulatory mechanisms employed by cis-encoded antisense RNAs. , 2007, Current opinion in microbiology.

[24]  Kay Nieselt,et al.  Global Transcriptional Start Site Mapping Using Differential RNA Sequencing Reveals Novel Antisense RNAs in Escherichia coli , 2014, Journal of bacteriology.

[25]  E. Gerhart H. Wagner,et al.  An Antisense RNA-Mediated Transcriptional Attenuation Mechanism Functions in Escherichia coli , 2002, Journal of bacteriology.

[26]  W. Mcallister,et al.  In a head-on collision, two RNA polymerases approaching one another on the same DNA may pass by one another. , 2009, Journal of molecular biology.

[27]  P. Deininger.  Molecular cloning: A laboratory manual , 1990 .

[28]  J. Collins,et al.  Tuning and controlling gene expression noise in synthetic gene networks , 2010, Nucleic acids research.

[29]  B. Voß,et al.  Evidence for a major role of antisense RNAs in cyanobacterial gene regulation , 2009, Molecular systems biology.

[30]  G. Fink,et al.  Antisense Transcription Controls Cell Fate in Saccharomyces cerevisiae , 2006, Cell.

[31]  O. Sliusarenko,et al.  Spatial organization of the flow of genetic information in bacteria , 2010, Nature.

[32]  S. French,et al.  Consequences of replication fork movement through transcription units in vivo. , 1992, Science.

[33]  Vitaly Epshtein,et al.  Transcription through the roadblocks: the role of RNA polymerase cooperation , 2003, The EMBO journal.

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

[35]  C. Rivetti,et al.  Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy , 2006, Nucleic acids research.

[36]  G. Church,et al.  Large-scale de novo DNA synthesis: technologies and applications , 2014, Nature Methods.

[37]  Z. Yakhini,et al.  Inferring gene regulatory logic from high-throughput measurements of thousands of systematically designed promoters , 2012, Nature Biotechnology.

[38]  R. Sorek,et al.  The excludon: a new concept in bacterial antisense RNA-mediated gene regulation , 2012, Nature Reviews Microbiology.

[39]  Adam P Arkin,et al.  Supplementary information for Rationally designed families of orthogonal RNA regulators of translation , 2012 .

[40]  O. Ozoline,et al.  Antisense transcription within the hns locus of Escherichia coli , 2010, Molecular Biology.

[41]  Eran Segal,et al.  Manipulating nucleosome disfavoring sequences allows fine-tune regulation of gene expression in yeast , 2012, Nature Genetics.

[42]  Narendra Maheshri,et al.  A regulatory role for repeated decoy transcription factor binding sites in target gene expression , 2012, Molecular systems biology.

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

[44]  J. Wade,et al.  Pervasive transcription: illuminating the dark matter of bacterial transcriptomes , 2014, Nature Reviews Microbiology.

[45]  Vivek K. Mutalik,et al.  Composability of regulatory sequences controlling transcription and translation in Escherichia coli , 2013, Proceedings of the National Academy of Sciences.

[46]  K. Shearwin,et al.  Transcriptional interference--a crash course. , 2005, Trends in genetics : TIG.

[47]  Herbert M Sauro,et al.  Visualization of evolutionary stability dynamics and competitive fitness of Escherichia coli engineered with randomized multigene circuits. , 2013, ACS synthetic biology.

[48]  R. Weiss,et al.  Directed evolution of a genetic circuit , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[49]  Erez Y. Levanon,et al.  Widespread occurrence of antisense transcription in the human genome , 2003, Nature Biotechnology.

[50]  W. Hess,et al.  cis-Antisense RNA, Another Level of Gene Regulation in Bacteria , 2011, Microbiology and Molecular Reviews.

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

[52]  Wu Wei,et al.  RNA Polymerase II Collision Interrupts Convergent Transcription , 2012, Molecular cell.

[53]  Kim Sneppen,et al.  A mathematical model for transcriptional interference by RNA polymerase traffic in Escherichia coli. , 2005, Journal of molecular biology.

[54]  Christopher A. Voigt,et al.  Characterization of 582 natural and synthetic terminators and quantification of their design constraints , 2013, Nature Methods.

[55]  G. Dougan,et al.  Cooperation Between Translating Ribosomes and RNA Polymerase in Transcription Elongation , 2010, Science.

[56]  Jeffrey W. Roberts,et al.  Mfd, the bacterial transcription repair coupling factor: translocation, repair and termination. , 2004, Current opinion in microbiology.

[57]  K. Jensen,et al.  The RNA chain elongation rate in Escherichia coli depends on the growth rate , 1994, Journal of bacteriology.

[58]  P. Cotter,et al.  Evidence for phenotypic bistability resulting from transcriptional interference of bvgAS in Bordetella bronchiseptica , 2013, Molecular microbiology.

[59]  Sriram Kosuri,et al.  Causes and Effects of N-Terminal Codon Bias in Bacterial Genes , 2013, Science.

[60]  Kristin Reiche,et al.  The primary transcriptome of the major human pathogen Helicobacter pylori , 2010, Nature.

[61]  Ian B. Dodd,et al.  Potent transcriptional interference by pausing of RNA polymerases over a downstream promoter. , 2009, Molecular cell.

[62]  Nicolas E. Buchler,et al.  Protein sequestration generates a flexible ultrasensitive response in a genetic network , 2009, Molecular systems biology.

[63]  Chien-Chung Chen,et al.  Genome Organization: The Effects of Transcription-driven DNA Supercoiling on Gene Expression Regulation , 2006 .

[64]  K. Kinzler,et al.  The Antisense Transcriptomes of Human Cells , 2008, Science.

[65]  J. Park,et al.  Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs , 2013, Nature Biotechnology.

[66]  E. Groisman,et al.  An antisense RNA that governs the expression kinetics of a multifunctional virulence gene , 2010, Molecular microbiology.

[67]  Christopher A. Voigt,et al.  Genomic Mining of Prokaryotic Repressors for Orthogonal Logic Gates , 2013, Nature chemical biology.

[68]  Katherine H. Huang,et al.  Operon formation is driven by co-regulation and not by horizontal gene transfer. , 2005, Genome research.

[69]  G. Church,et al.  RNA expression analysis using a 30 base pair resolution Escherichia coli genome array , 2000, Nature Biotechnology.

[70]  B. Simmons,et al.  A single-base resolution map of an archaeal transcriptome. , 2010, Genome research.

[71]  M. Palumbo,et al.  Widespread Antisense Transcription in Escherichia coli , 2010, mBio.

[72]  Gábor Balázsi,et al.  Transferring a synthetic gene circuit from yeast to mammalian cells , 2013, Nature Communications.

[73]  Daniel B. Sloan,et al.  Antisense Transcription Is Pervasive but Rarely Conserved in Enteric Bacteria , 2012, mBio.

[74]  Jun Ma.  Gene Expression and Regulation , 2006 .

[75]  K. Shearwin,et al.  Transcriptional interference between convergent promoters caused by elongation over the promoter. , 2004, Molecular cell.

[76]  Konstantin S. Shavkunov,et al.  Intragenic PROMOTOR-Like Sites in the genome of Escherichia coli Discovery and Functional Implication , 2007, J. Bioinform. Comput. Biol..

[77]  T. Gingeras,et al.  Genome-wide antisense transcription drives mRNA processing in bacteria , 2011, Proceedings of the National Academy of Sciences.

[78]  Evgeny Nudler,et al.  RNA Polymerase Backtracking in Gene Regulation and Genome Instability , 2012, Cell.

[79]  Jason T Stevens,et al.  Designing RNA-based genetic control systems for efficient production from engineered metabolic pathways. , 2015, ACS synthetic biology.

[80]  G. Storz,et al.  Bacterial antisense RNAs: how many are there, and what are they doing? , 2010, Annual review of genetics.

[81]  G. Church,et al.  Accurate multiplex gene synthesis from programmable DNA microchips , 2004, Nature.

[82]  J. Wade,et al.  Widespread suppression of intragenic transcription initiation by H-NS , 2014, Genes & development.

[83]  B. Dujon.  The yeast genome project: what did we learn? , 1996, Trends in genetics : TIG.

[84]  Vitaly Epshtein,et al.  Cooperation Between RNA Polymerase Molecules in Transcription Elongation , 2003, Science.

[85]  Christopher A. Voigt,et al.  Principles of genetic circuit design , 2014, Nature Methods.

[86]  Rho Factor: Transcription Termination in Four Steps , 2003, Current Biology.

[87]  Yaoping Liu and Ichizo Kobayashi with Intragenic Reverse Promoters Restriction Enzyme Gene Is Associated Negative Regulation of the EcoRI , 2007 .

[88]  R. Landick,et al.  Rho and NusG suppress pervasive antisense transcription in Escherichia coli. , 2012, Genes & development.

[89]  B. Schwikowski,et al.  Condition-Dependent Transcriptome Reveals High-Level Regulatory Architecture in Bacillus subtilis , 2012, Science.

[90]  B. Michel,et al.  Transcription‐induced deletions in Escherichia coli plasmids , 1995, Molecular microbiology.

[91]  C. O'Connor,et al.  Highly repressible expression system for cloning genes that specify potentially toxic proteins , 1987, Journal of bacteriology.

[92]  E. Greenberg,et al.  Antisense RNA that affects Rhodopseudomonas palustris quorum-sensing signal receptor expression , 2012, Proceedings of the National Academy of Sciences.

[93]  Christopher A. Voigt,et al.  Advances in genetic circuit design: novel biochemistries, deep part mining, and precision gene expression. , 2013, Current opinion in chemical biology.

[94]  Christopher A. Voigt,et al.  Ribozyme-based insulator parts buffer synthetic circuits from genetic context , 2012, Nature Biotechnology.

[95]  Bronwyn G. Butcher,et al.  Transcriptome Analysis of Pseudomonas syringae Identifies New Genes, Noncoding RNAs, and Antisense Activity , 2010, Journal of bacteriology.

[96]  Wei-Shou Hu,et al.  Convergent Transcription in the Butyrolactone Regulon in Streptomyces coelicolor Confers a Bistable Genetic Switch for Antibiotic Biosynthesis , 2011, PloS one.