Riboswitches in unexpected places--a synthetic riboswitch in a protein coding region

In natural and engineered systems, cis-RNA regulatory elements such as riboswitches are typically found within untranslated regions rather than within the protein coding sequences of genes. However, RNA sequences with important regulatory roles can exist within translated regions. Here, we present a synthetic riboswitch that is encoded within the translated region of a gene and represses Escherichia coli gene expression greater than 25-fold in the presence of a small-molecule ligand. The ability to encode riboswitches within translated regions as well as untranslated regions provides additional opportunities for creating new genetic control elements. Furthermore, evidence that a riboswitch can function in the translated region of a gene suggests that future efforts to identify natural riboswitches should consider this possibility.     

Common themes and differences in SAM recognition among SAM riboswitches

The recent discovery of short cis-acting RNA elements termed riboswitches has caused a paradigm shift in our understanding of genetic regulatory mechanisms. The three distinct superfamilies of S-adenosyl-l-methionine (SAM) riboswitches are the most commonly found riboswitch classes in nature. These RNAs represent three independent evolutionary solutions to achieve specific SAM recognition. This review summarizes research on 1) modes of gene regulatory mechanisms, 2) common themes and differences in ligand recognition, and 3) ligand-induced conformational dynamics among SAM riboswitch families. The body of work on the SAM riboswitch families constitutes a useful primer to the topic of gene regulatory RNAs as a whole.     

Conformational capture of the SAM-II riboswitch

Riboswitches are gene regulation elements in mRNA that function by specifically responding to metabolites. Although the metabolite-bound states of riboswitches have proven amenable to structure determination efforts, knowledge of the structural features of riboswitches in their ligand-free forms and their ligand-response mechanisms giving rise to regulatory control is lacking. Here we explore the ligand-induced folding process of the S-adenosylmethionine type II (SAM-II) riboswitch using chemical and biophysical methods, including NMR and fluorescence spectroscopy, and single-molecule fluorescence imaging. The data reveal that the unliganded SAM-II riboswitch is dynamic in nature, in that its stem-loop element becomes engaged in a pseudoknot fold through base-pairing with nucleosides in the 3' overhang containing the Shine-Dalgarno sequence. Although the pseudoknot structure is highly transient in the absence of its ligand, S-adenosylmethionine (SAM), it becomes conformationally restrained upon ligand recognition, through a conformational capture mechanism. These insights provide a molecular understanding of riboswitch dynamics that shed new light on the mechanism of riboswitch-mediated translational regulation.     

Enzymatic probing analysis of an engineered riboswitch reveals multiple off conformations

We investigated the gene regulatory mechanism of a previously engineered riboswitch +thiMN(15)#19 that turns on gene expression in response to thiamine pyrophosphate (TPP). In vitro enzymatic probing was performed to identify the secondary structures of the OFF conformations predicted by Mfold. Interestingly, enzymatic probing data of the riboswitch and its variants indicated that the riboswitch in its OFF state adopts two distinct structures. Moreover, further in vivo experiments suggested that both OFF structures contribute to the riboswitch function. A deeper understanding of how riboswitches function at the molecular level should enhance our ability to design synthetic riboswitches with new or improved characteristics.     

Enzymatic Probing Analysis of an Engineered Riboswitch Reveals Multiple off Conformations

We investigated the gene regulatory mechanism of a previously engineered riboswitch +thiMN₁₅#19 that turns on gene expression in response to thiamine pyrophosphate (TPP). In vitro enzymatic probing was performed to identify the secondary structures of the OFF conformations predicted by Mfold. Interestingly, enzymatic probing data of the riboswitch and its variants indicated that the riboswitch in its OFF state adopts two distinct structures. Moreover, further in vivo experiments suggested that both OFF structures contribute to the riboswitch function. A deeper understanding of how riboswitches function at the molecular level should enhance our ability to design synthetic riboswitches with new or improved characteristics.     

Single-molecule force spectroscopy of the add adenine riboswitch relates folding to regulatory mechanism

Riboswitches regulate gene expression via ligand binding to an aptamer domain which induces conformational changes in a regulatory expression platform. By unfolding and refolding single add adenine riboswitch molecules in an optical trap, an integrated picture of the folding was developed and related to the regulatory mechanism. Force-extension curves (FECs) and constant-force folding trajectories measured on the aptamer alone revealed multiple partially-folded states, including several misfolded states not on the native folding pathway. All states were correlated to key structural components and interactions within hierarchical folding pathways. FECs of the full-length riboswitch revealed that the thermodynamically stable conformation switches upon ligand binding from a structure repressing translation to one permitting it. Along with rapid equilibration of the two structures in the absence of adenine, these results support a thermodynamically-controlled regulatory mechanism, in contrast with the kinetic control of the closely-related pbuE adenine riboswitch. Comparison of the folding of these riboswitches revealed many similarities arising from shared structural features but also essential differences related to their different regulatory mechanisms.     

Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain

Riboswitches are cis-acting genetic regulatory elements found commonly in bacterial mRNAs that consist of a metabolite-responsive aptamer domain coupled to a regulatory switch. Purine riboswitches respond to intracellular concentrations of either adenine or guanine/hypoxanthine to control gene expression. The aptamer domain of the purine riboswitch contains a pyrimidine residue (Y74) that forms a Watson-Crick base-pairing interaction with the bound purine nucleobase ligand that discriminates between adenine and guanine. We sought to understand the structural basis of this specificity and the mechanism of ligand recognition by the purine riboswitch. Here, we present the 2,6-diaminopurine-bound structure of a C74U mutant of the xpt-pbuX guanine riboswitch, along with a detailed thermodynamic and kinetic analysis of nucleobase recognition by both the native and mutant riboswitches. These studies demonstrate clearly that the pyrimidine at position 74 is the sole determinant of purine riboswitch specificity. In addition, the mutant riboswitch binds adenine and adenine derivatives well compared with the guanine-responsive riboswitch. Under our experimental conditions, 2,6-diaminopurine binds the RNA with DeltaH=-40.3 kcal mol(-1), DeltaS=-97.6 cal mol(-1)K(-1), and DeltaG=-10.73 kcal mol(-1). A kinetic determination of the slow rate (0.15 x 10(5)M(-1)s(-1) and 2.1 x 10(5)mM(-1)s(-1) for 2-aminopurine binding the adenine-responsive mutant riboswitch and 7-deazaguanine-binding guanine riboswitch, respectively) of association under varying experimental conditions allowed us to propose a mechanism for ligand recognition by the purine riboswitch. A conformationally dynamic unliganded state for the binding pocket is stabilized first by the Watson-Crick base pairing between the ligand and Y74, and by the subsequent ordering of the J2/3 loop, enclosing the ligand within the three-way junction.     

Themes and variations in riboswitch structure and function

The complexity of gene expression control by non-coding RNA has been highlighted by the recent progress in the field of riboswitches. Discovered a decade ago, riboswitches represent a diverse group of non-coding mRNA regions that possess a unique ability to directly sense cellular metabolites and modulate gene expression through formation of alternative metabolite-free and metabolite-bound conformations. Such protein-free metabolite sensing domains utilize sophisticated three-dimensional folding of RNA molecules to discriminate between a cognate ligand from related compounds so that only the right ligand would trigger a genetic response. Given the variety of riboswitch ligands ranging from small cations to large coenzymes, riboswitches adopt a great diversity of structures. Although many riboswitches share structural principles to build metabolite-competent folds, form precise ligand-binding pockets, and communicate a ligand-binding event to downstream regulatory regions, virtually all riboswitch classes possess unique features for ligand recognition, even those tuned to recognize the same metabolites. Here we present an overview of the biochemical and structural research on riboswitches with a major focus on common principles and individual characteristics adopted by these regulatory RNA elements during evolution to specifically target small molecules and exert genetic responses. This article is part of a Special Issue entitled: Riboswitches.     

Single-molecule studies of riboswitch folding

The folding dynamics of riboswitches are central to their ability to modulate gene expression in response to environmental cues. In most cases, a structural competition between the formation of a ligand-binding aptamer and an expression platform (or some other competing off-state) determines the regulatory outcome. Here, we review single-molecule studies of riboswitch folding and function, predominantly carried out using single-molecule FRET or optical trapping approaches. Recent results have supplied new insights into riboswitch folding energy landscapes, the mechanisms of ligand binding, the roles played by divalent ions, the applicability of hierarchical folding models, and kinetic vs. thermodynamic control schemes. We anticipate that future work, based on improved data sets and potentially combining multiple experimental techniques, will enable the development of more complete models for complex RNA folding processes. This article is part of a Special Issue entitled: Riboswitches.     

Riboswitch regulation mechanisms: RNA,metabolites and regulatory proteins

Riboswitches are RNA sensors that have been shown to modulate the expression of downstream genes by altering their structure upon metabolite binding. Riboswitches are unique among cellular regulators in that metabolite detection is strictly performed using RNA interactions with the sensed metabolite and in which no regulatory protein is needed to mediate the interaction. However, recent studies have shed light on riboswitch control mechanisms relying on protein regulators to harness metabolite binding for the mediation of gene expression, thereby increasing the range of cellular factors involved in riboswitch regulation. The interaction between riboswitches and proteins adds another level of evolutionary pressure as riboswitches must maintain key residues for metabolite detection, structural switching and protein binding sites. Here, we review regulatory mechanisms involving Escherichia coli riboswitches that have recently been shown to rely on regulatory proteins. We also discuss the implication of such protein-based riboswitch regulatory mechanisms for genetic regulation.     

High-throughput screening of cell-free riboswitches by fluorescence-activated droplet sorting

Cell-free systems that display complex functions without using living cells are emerging as new platforms to test our understanding of biological systems as well as for practical applications such as biosensors and biomanufacturing. Those that use cell-free protein synthesis (CFPS) systems to enable genetically programmed protein synthesis have relied on genetic regulatory components found or engineered in living cells. However, biological constraints such as cell permeability, metabolic stability, and toxicity of signaling molecules prevent development of cell-free devices using living cells even if cell-free systems are not subject to such constraints. Efforts to engineer regulatory components directly in CFPS systems thus far have been based on low-throughput experimental approaches, limiting the availability of basic components to build cell-free systems with diverse functions. Here, we report a high-throughput screening method to engineer cell-free riboswitches that respond to small molecules. Droplet-sorting of riboswitch variants in a CFPS system rapidly identified cell-free riboswitches that respond to compounds that are not amenable to bacterial screening methods. Finally, we used a histamine riboswitch to demonstrate chemical communication between cell-sized droplets.     

A flow cytometry-based screen for synthetic riboswitches

Riboswitches regulate gene expression through direct, small molecule–mRNA interactions. The creation of new synthetic riboswitches from in vitro selected aptamers benefits from rapid, high-throughput methods for identifying switches capable of triggering dramatic changes in gene expression in the presence of a desired ligand. Here we present a flow cytometry-based screen for identifying synthetic riboswitches that induce robust increases in gene expression in the presence of theophylline. The performance characteristics of our newly identified riboswitches exceed those of previously described natural and synthetic riboswitches. Sequencing data and structure probing experiments reveal the ribosome binding site to be an important determinant of how well a switch performs and may provide insights into the design of new synthetic riboswitches.     

Computational analysis of riboswitch-based regulation

Advances in computational analysis of riboswitches in the last decade have contributed greatly to our understanding of riboswitch regulatory roles and mechanisms. Riboswitches were originally discovered as part of the sequence analysis of the 5′-untranslated region of mRNAs in the hope of finding novel gene regulatory sites, and the existence of structural RNAs appeared to be a spurious phenomenon. As more riboswitches were discovered, they illustrated the diversity and adaptability of these RNA regulatory sequences. The fact that a chemically monotonous molecule like RNA can discern a wide range of substrates and exert a variety of regulatory mechanisms was subsequently demonstrated in diverse genomes and has hastened the development of sophisticated algorithms for their analysis and prediction. In this review, we focus on some of the computational tools for riboswitch detection and secondary structure prediction. The study of this simple yet efficient form of gene regulation promises to provide a more complete picture of a world that RNA once dominated and allows rational design of artificial riboswitches. This article is part of a Special Issue entitled: Riboswitches.