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<p style="font-size:20px"><b>Riboswitches: gene expression regulating devices</b></p>

<p>Riboswitches are regulatory domains present in messenger RNA (mRNA) molecules that can bind to specific molecules, or ligands, thereby enhancing or preventing the production of encoded proteins. RNA is a good platform for these responsive motifs not only due to its central role in gene expression and protein production, but also given its increased structural flexibility, thereby accommodating various conformations and conformational changes that enable the recognition of a wide range of regulatory signals. Even though there are several types of riboswitches, each underpinned by different mechanisms of activity, they can be generally described as nucleic acid secondary structures that, upon exposure to a broad range of stimuli (e.g. amino acids, metal ions, other nucleic acids, changes in temperature and/or pH), sustain a conformational change that physically enables or prevents protein production (Garst et al., 2011). Broadly, riboswitches can be identified as part of two regulatory motifs, where some of them have activity at the transcriptional level and the remainder act at the translational level.  </p>

<p>They were first discovered in bacteria in 2002 as RNA-based devices with intracellular sensing capabilities holding affinity for vitamin derivatives (Winkler et al., 2002a, Winkler et al. 2002b). More generally, they have been identified in both Gram-positive and Gram-negative bacteria, where they serve as intramolecular switches  that respond to the binding of structurally-diverse metabolites. To date, there have been nearly 40 different classes of riboswitches reported, modeled (with atomic resolution in complex with their ligands), and experimentally validated. These research findings have revealed that riboswitches, as non-coding RNA domains, exploit several distinct structural and conformational features to produce binding pockets with very high selectivity for their ligands. In fact, they have been shown to control a broad range of genes across bacterial species, ranging from those involved in metabolism to the uptake of amino acids, co-factors, nucleotides, and metal ions. Moreover, their activity can be regulated not only via coupling them to their ligand, but also through other nucleic-acid relevant parameters such as temperature and ionic strength. </p>

<p>Transcription-regulating riboswitches control the production of their own mRNA molecules. In this case, the riboswitch - encoded upstream - is transcribed first and self-assembles into its functional secondary structure, which depending on the biochemical or environmental conditions it responds to, determines whether the process will continue downstream (Blouin et al., 2009). For instance, an OFF state riboswitch, when exposed to its target ligand, will have a secondary structure in the form of a terminator, thereby stopping the RNA polymerase from transcribing the remainder of the mRNA. Conversely, when the riboswitch is in an ON state, the anti-terminator secondary structure is adopted allowing the polymerase to produce the full transcript. In that sense, riboswitches have functionality in cis to control protein expression within the transcript they are encoded in, distingushing them from other small RNA molecules that act in trans in regulate other messenger RNAs. </p>

<p>On the other hand, riboswitches that act at the translational level usually sequester the Ribosome Binding Site (RBS) within their secondary structure to physically prevent the ribosome from binding and translating the transcript, as shown graphically in Fig. 3. Thus, responsiveness to stimuli in this scenario is provided by controllably providing access to the RBS via a number of mechanisms. For instance, cleavage-mediated activation takes place when the secondary structure of the riboswitch, as determined by their target, reveals an enzyme restriction site. Once the riboswitch is cleaved, the ribosome can bind to the RBS and undertake protein production. Similarly, other triggering mechanisms include ligand-induced changes in conformation, where the RBS is secluded or released within/from a hairpin structure, enabling translation through the now-accessible ribosome binding domain. Examples of ligands include other RNA molecules which carry a complementary domain to a toehold that upon binding opens the hairpin and exposes the RBS to mediate translation. This type of translation-regulating motifs, known as toehold switches (Green et al., 2014), have found application in a number of genetic and nucleic acid circuitry within synthetic biological systems.  </p>

[[File:FNA riboswitch.png|500px|center|thumb| <b>Figure 3: Theophylline-responsive riboswitch.</b> The Ribosome Binding Site (RBS) is part of the stem of a hairpin, thus becoming inaccessible to the ribosome and inhibiting protein translation. The presence of a theophylline recognition site within the stem induces a conformational change upon theophylline binding, which in turn exposes the RBS and makes it accessible for the ribosome to initiate translation.]]


<p style="font-size:14px"><b>References</b></p>
<p>Garst, A., Edwards, A.L., & Batey, R.T. (2011). Riboswitches: Structures and Mechanisms. Cold Spring Harb Perspect Biol, a003533. https://doi.org/10.1101/cshperspect.a003533</p>
<p>Winkler, W., Nahvi, A. & Breaker, R. (2002) Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419, 952–956. https://doi.org/10.1038/nature011459</p>
<p>Winkler, W., Cohen-Chalamish, S., Breaker, R. (2002) A mRNA structure that controls gene expression by binding FMN. Proceedings of the National Academy of Sciences of the United States of America 99 (25) 15908-15913. https://doi.org/10.1073/pnas.212628899 </p>
<p>Blouin, S., Mulhbacher, J., Penedo, J.C., & Lafontaine, D. A. (2009). Riboswitches: Ancient and Promising Genetic Regulators. ChemBioComm 10, 400-416. https://doi.org/10.1002/cbic.200800593</p>
<p>Green, A. A, Silver, P.A., Collins, J.J., and Peng,Y. (2014). Toehold Switches: De-novo-Designed Regulators of Gene Expression. Cell 159(4) 925-939. https://doi.org/10.1016/j.cell.2014.10.002</p>