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Letter
Transcriptional Interference in convergent promoters as a means for Tunable Gene Expression Antoni Escalas Bordoy, Usha S. Varanasi, Colleen M Courtney, and Anushree Chatterjee ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00223 • Publication Date (Web): 26 Jun 2016 Downloaded from http://pubs.acs.org on June 28, 2016
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Transcriptional Interference in convergent promoters as a means for Tunable Gene Expression
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Antoni E. Bordoy1, Usha S. Varanasi1, Colleen M. Courtney1 and Anushree Chatterjee1,2,*
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Boulder, 3415 Colorado Avenue, UCB 596, CO 80303, USA.
Department of Chemical and Biological Engineering, 2BioFrontiers institute, University of Colorado,
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ABSTRACT: An important goal of synthetic biology involves the extension and standardization of
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novel biological elements for applications in medicine and biotechnology. Transcriptional
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interference, occurring in sets of convergent promoters, offers a promising mechanism for building
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elements for the design of tunable gene regulation. Here, we investigate the transcriptional
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interference mechanisms of antisense roadblock and RNA polymerase traffic in a set of convergent
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promoters as novel modules for synthetic biology. We show examples of elements, including
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antisense roadblock, relative promoter strengths, inter-promoter distance, and sequence content
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that can be tuned to give rise to repressive as well as cooperative behaviors, therefore resulting in
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distinct gene expression patterns. Our approach will be useful towards engineering new biological
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devices and will bring new insights to naturally occurring cis-antisense systems. Therefore, we are
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reporting a new biological tool that can be used for synthetic biology.
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KEY WORDS: Antisense transcription, transcriptional interference, gene regulation, antisense
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roadblock, genetic switches, RNA polymerase collision.
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Rational design of new biological systems that allow for better gene regulation has been of
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outstanding interest in synthetic biology. Their various purposes are to improve biosensing, biofuel and
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pharmaceutical production, and develop gene therapies that target superbugs and diseases like cancer1.
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The first synthetic genetic devices designed to improve the control of gene expression, the toggle switch2
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and repressilator3, paved the way for more complex transcriptional networks interconnected by different
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repressor/activator-promoter architectures that enabled construction of genetic switches4,5, oscillators6,
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memory elements7, logic gates7,8, RNA devices9,10 and quorum sensing systems11,12. To achieve such
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complex cellular responses a vast range of orthogonal regulatory parts have been rationally designed
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including promoters13, proteins14 and riboregulators15. Despite such great advancements, most of synthetic
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biology devices that implement regulation via proteins, RNA or both16, consist of separated,
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interconnected transcriptional units with trans-acting elements, thus requiring coordination between
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multiple biological parts often leading to high metabolic costs17. Here, inspired by naturally occurring
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systems, we report a new category of synthetic biological devices that are connected and regulate gene
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expression through the process of transcriptional interference during cis-antisense transcription. Since this
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form of regulation occurs during the process of transcription, it reduces the time scale of the regulatory
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response upon a certain stimulus and lowers the associated cellular energetic cost.
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Cis-antisense transcription arises when a pair of promoters is arranged in convergent orientation
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(one in the sense orientation and one in the antisense orientation) via a mechanism termed transcriptional
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interference18 (TI) that has been observed to play key regulatory role in both prokaryotes19–22 and
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eukaryotes23–26. Presence of TI (reviewed in ref. 28) at various promoter arrangements has been shown to
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be ubiquitous20,27–29 and it is therefore believed to be conserved over evolution30. Additionally,
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cis-antisense transcription allows for significant DNA compression, i.e. it reduces the genomic footprint31.
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Previously we have shown that cis-antisense transcription has a built-in regulatory role in conferring
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biological systems with robust bistable genetic switch-like characteristics19,22,32,33. Despite the widespread
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occurrence of such promoter arrangements, little effort has gone into taking advantage of its regulatory
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mechanisms for designing synthetic biology devices. Only recently, the CRISPR-dCas9 system has been
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used to create genetic switches using the TI mechanism of roadblock34. Here, to the best of our
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knowledge, we present the first attempt to use synthetic TI constructs to understand how the different
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mechanisms of TI can be integrated to achieve regulation of gene expression. In order to do so, we take
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advantage of the TI mechanisms of (i) Roadblock, where a DNA bound protein blocks movement of an
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elongating RNA polymerase (RNAP)35 (Fig. 1a) and (ii) RNAP traffic, that can either increase the local
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RNAP concentration at the convergent promoters, or cause RNAP collision in which two opposing
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elongation complexes (ECs) collide while traversing the overlapping DNA between two convergent
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promoters often producing truncated transcripts19 (Fig. 1b). Collision between two opposing ECs is
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influenced by build-up of torsional stress during movement of a transcribing EC on DNA, which is
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preceded by positive super-coiling and followed by negative super-coiling36, resulting in either RNAP
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stalling or RNAP backtracking37,38. Although it has been shown that transcription bubbles can pass by one
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another39, there is sufficient evidence that approach of two convergent ECs causes RNAP to fall off the
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DNA with consequent production of truncated RNAs19. Since both roadblock and collision affect RNAP
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traffic over the shared DNA between the convergent promoters, unraveling the nature of these forms of
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regulation will enable us to use these new transcription-based regulated devices as powerful tools for both
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scientific research and biotechnology. We show that experimental parameters including antisense
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roadblock, relative promoter strengths, inter-promoter distance, and RNAP residence time in the
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overlapping DNA can be used to build distinct expression patterns both in sense and antisense genes.
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Inspired by naturally occurring systems, we arranged two biological components: (i) inducible
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synthetic PLTet and PLLac promoters regulated by repressor proteins TetR and LacI40, respectively, and
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(ii) overlapping DNA of various lengths containing naturally occurring bacterial sequences41–45,
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separating PLTet and PLLac, in order to construct sets of convergent promoters to investigate effect of RNAP
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traffic and roadblock (Fig. 2a, Supporting Text 1, Table S1-S2). In our experiments, PLTet and PLLac drive
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expression of green fluorescent protein (GFP) and red fluorescent protein (mCherry), respectively, which
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provide a visible read out of sense-antisense gene expression. Although the use sense and antisense terms
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is relative to both strands of DNA, hereafter we refer to sense and antisense strand as the ones that contain
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PLtet and PLLac promoters, respectively. Various plasmid constructs were harbored by four different strains
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of model bacterial organism E. coli that expressed either both TetR and LacI repressors (DH5αZ1), only
RESULTS AND DISCUSSION
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TetR (DH5α-TetR), only LacI (C600Zi), or none of the repressors (DH5α-WT), in order to study extreme
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cases of promoter activity depending on the repressors expressed (Fig. 2b, Fig. S1). The transcriptional
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rates of the convergent, orthogonal PLTet and PLLac promoters were changed by inducing them with
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different levels of anhydrous tetracycline (aTc) and Isopropyl β-D-1-thiogalactopyranoside (IPTG),
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respectively. Single sense and antisense promoter constructs driving expression of respective reporter
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genes were designed to serve as controls (Fig. 2c, d, Supporting Text 1). Upon addition of only one
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inducer, one promoter becomes active while the other remains inactive. We hypothesize activity of the
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induced promoter can be regulated by the TI mechanism of antisense roadblock whereby the neighboring
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repressor protein, which is bound to the DNA, impedes the movement of the transcribing RNAP that, in
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some cases, leads to failed transcription35. We further hypothesize that the sequence content of the
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overlapping DNA between the convergent promoters can be used as a tunable synthetic biology module,
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to control gene expression to build various types of genetic switches. Thus, here we present various
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examples of systems where tunability of gene expression can be achieved due to transcriptional processes.
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During convergent transcription both sense and antisense transcripts share complementary regions
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that can potentially participate in RNA interactions. To confirm that the mechanisms being investigated
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are related to TI and to rule out role antisense RNA interactions, we measured protein expression in a
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dual-plasmid system containing sense construct, pAE_T145 expressing sense RNA fused to GFP
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transcript, and the corresponding antisense construct with the complementary overlapping DNA of the
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same length, pAE_L145_R expressing antisense RNA fused to mCherry transcript in trans (Supporting
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Text 1, Fig. S1), in strain C600Zi (Supporting Text 2) at various IPTG concentrations. We observed
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significant changes in mCherry expression at both 0.05 and 0.5 mM IPTG (p-value
