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Small-Molecule-Binding Riboswitches THEA S Small-Molecule-Binding Riboswitches THEA S. LOTZ1 and BEATRIX SUESS1 1Synthetic Genetic Circuits, Department of Biology, TU Darmstadt, 64287 Darmstadt, Germany ABSTRACT RNA is a versatile biomolecule capable of moieties is imperative in enabling RNA to function as a transferring information, taking on distinct three-dimensional biological catalyst, regulator, or structural scaffold. shapes, and reacting to ambient conditions. RNA molecules In the last 15 years, many different types of regula- utilize a wide range of mechanisms to control gene expression. tory RNA molecules have been discovered in nature. An An example of such regulation is riboswitches. Consisting exclusively of RNA, they are able to control important metabolic abundance of riboswitches, ribozymes, RNA thermom- processes, thus providing an elegant and efficient RNA-only eters, and short and long noncoding RNAs have been regulation system. Existing across all domains of life, found in all three domains of life. However, plenty of riboswitches appear to represent one of the most highly questions regarding how RNA influences cell physio- conserved mechanisms for the regulation of a broad range of logy, differentiation, and development still remain un- biochemical pathways. Through binding of a wide range of answered and the subject of extensive research (1). There small-molecule ligands to their so-called aptamer domain, are several advantages when gene expression is regulated riboswitches undergo a conformational change in their downstream “expression platform.” In consequence, the pattern via RNA alone as opposed to regulation in combination of gene expression changes, which in turn results in increased or with proteins. It allows (i) faster regulatory responses, decreased protein production. Riboswitches unite the sensing (ii) easier transfer of a single-step genetic control element and transduction of a signal that can directly be coupled to to other organisms, and (iii) flexible combination with the metabolism of the cell; thus they constitute a very potent different downstream readout platforms for a maximum regulatory mechanism for many organisms. Highly specific of regulatory outputs (2). in vivo RNA-binding domains not only occur but can also be Riboswitches are protein-independent, RNA-based evolved by means of the SELEX (systematic evolution of ligands — by exponential enrichment) method, which allows in vitro gene regulatory elements short, structured RNA ele- selection of aptamers against almost any ligand. Coupling of ments able to regulate gene expression in response to these aptamers with an expression platform has led to the binding a small-molecule ligand. Their use allows tem- development of synthetic riboswitches, a highly active research poral and dosage control over gene expression. Follow- field of great relevance and immense potential. The aim of this ing the discovery and validation of the first riboswitch in review is to summarize developments in the riboswitch field over 2002 (3), they have been the subject of intense research. the last decade and address key questions of recent research. Received: 11 February 2018, Accepted: 11 May 2018, INTRODUCTION Published: 3 August 2018 Editors: Gisela Storz, Division of Molecular and Cellular Biology, Traditionally, the functional role of RNA was thought Eunice Kennedy Shriver National Institute of Child Health and to be restricted to transferring genetic information Human Development, Bethesda, MD; Kai Papenfort, Department of Biology I, Microbiology, LMU Munich, Martinsried, Germany from DNA to protein. However, the discovery of RNA Citation: Lotz TS, Suess B. 2018. Small-molecule-binding elements mediating gene control, chemical reaction riboswitches. Microbiol Spectrum 6(4):RWR-0025-2018. catalysis, and signal transduction has changed this per- doi:10.1128/microbiolspec.RWR-0025-2018. ception fundamentally. Its ability to form complex three- Correspondence: Beatrix Suess, [email protected] © 2018 American Society for Microbiology. All rights reserved. dimensional structures that precisely present chemical ASMscience.org/MicrobiolSpectrum 1 Downloaded from www.asmscience.org by IP: 157.89.65.129 On: Wed, 08 Aug 2018 13:08:36 Lotz and Suess Their mechanism of action and importance for their typically a metabolite. They consist of two domains, host organism have been explored (4–6), along with the the so-called aptamer domain and the expression plat- generation of new, synthetic riboswitches by genetic form. The aptamer domain selectively recognizes its engineers (7, 8). small-molecule ligand while at the same time discrimi- In this review, we have compiled information on all nating against closely related variants of the ligand. the natural riboswitches discovered so far and discuss Binding of the ligand to the aptamer domain leads to their mechanisms, occurrence, and potential to develop structural changes in the following expression platform, genetic control elements for genetic analyses and syn- inducing altered expression of the downstream mRNA. thetic biology in the future. The changes in gene expression typically include regu- lating transcription termination or translation initiation (Fig. 1), more rarely, ribozyme-mediated mRNA degra- RIBOSWITCHES—LOCATION, MECHANISM, dation or the control of splicing. AND DISTRIBUTION Riboswitches are widely distributed throughout the Riboswitches are highly structured RNA sequence ele- bacterial world and are increasingly found in eukaryotes ments typically located in the 5′ untranslated region and archaea. They are divided into so-called classes, (5′ UTR) of many bacterial mRNAs that control a groups of riboswitches that respond to the same ligand plethora of metabolic processes. They act as molecular and that show a similar conserved core structure. There switches regulating gene expression via conformational can be more than one class of riboswitches binding changes in their three-dimensional structure upon direct to the same ligand. However, the classes then have dis- interaction (“binding”) with a specific small molecule, tinctly different core structures. For example, among FIGURE 1 Common mechanism of riboswitches in bacteria. (A) Regulation of translation initiation: In the absence of the ligand, a stem-loop structure is formed between the aptamer domain and a sequence complementary to the Shine-Dalgarno (SD) sequence. Thus, the SD sequence is accessible for 30S binding, and translation initiation occurs. As a consequence of ligand binding (pentagon) and the folding of the aptamer domain, an alternative stem-loop is formed, which sequesters the SD sequence, and the binding of the 30S ribosomal subunit is blocked. (B) Regulation of transcription termination: The aptamer domain is followed by a sequence complementary to the 3′ part of the aptamer and a U stretch. In the absence of the ligand, the complementary 3′ part is base-paired with the aptamer, forming a terminator structure. Thus, RNA polymerase (RNAP) dissociates and transcription is blocked. Upon ligand binding, terminator structure formation is inhibited and transcription can proceed, resulting in expression of the reporter gene. 2 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 157.89.65.129 On: Wed, 08 Aug 2018 13:08:36 Small-Molecule-Binding Riboswitches the prequeuosine-1 (preQ1) binding riboswitches, there system within an organism. This is concurrent with the are major differences between classes, as, e.g., preQ1-I now widely accepted RNA world theory, which pro- riboswitches are small (25 to 45 nucleotides long) poses a phase of evolution where there were no proteins whereas preQ1-II have a larger, more complex core and DNA and all their current roles were carried out by consensus sequence (average, 58 nucleotides). Between RNA on its own. Modern riboswitches could be descen- the two classes, there are no detectable structure or dants of these ancient regulatory systems (6, 27, 28). sequence similarities (9, 10). In the meantime, close to 40 riboswitch classes have been identified and many more await discovery. The variety of ligands ranges THE TPP RIBOSWITCH—AN EXAMPLE from vitamins, amino acids, and nucleotides to second ACROSS ALL DOMAINS OF LIFE messengers and a number of ions. TPP-binding riboswitches belong to the most widely Several excellent reviews about riboswitches have distributed riboswitch class. They are found in all do- been published in recent years (4, 11–13). For a com- mains of life; the structure of their aptamer domain is prehensive overview, we have compiled a table (Table 1) highly conserved. Important differences between the TPP summarizing all riboswitch classes described to date. In riboswitches can be found in their regulation mecha- addition to the recognized ligands, we list the commonly nisms. In bacteria, ligand binding to the aptamer domain regulated genes and the main organisms in which the leads to changed transcription termination and transla- riboswitches occur. In addition, we include the year of tion initiation, respectively. Occasionally, an organism discovery of the ligand-riboswitch pair and the first de- also uses both mechanisms. Escherichia coli, for exam- scription of a three-dimensional structure if available. ple, controls the thiM operon via a TPP riboswitch reg- Albeit many RNA motifs have been justly suspected to ulated by translation initiation, whereas the thiC operon act as riboswitches since their discovery (14, 15), some is regulated both at the translational and transcriptional “orphan riboswitches”
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