Riboswitches are gene regulation elements within RNA that recognize specific metabolites. They predominantly occur in the untranslated leader regions of bacterial messenger RNA (mRNA). Upon ...metabolite binding to the aptamer domain, a structural change in the adjoining downstream expression platform signals “on” or “off” for gene expression. Researchers have achieved much progress in characterizing ligand-bound riboswitch states at the molecular level; an impressive number of high-resolution structures of aptamer–ligand complexes is now available. These structures have significantly contributed toward our understanding of how riboswitches interact with their natural ligands and with structurally related analogues. In contrast, relatively little is known about the nature of the unbound (apo) form of riboswitches. Moreover, the details of how changes in the aptamer domain are transduced into conformational changes in the decision-making expression platform remain murky. In this Account, we report on recent efforts aimed at the characterization of free states, ligand recognition, and ligand-induced folding in riboswitches. Riboswitch action is best approached as a cotranscriptional process, which implies sequential folding and release of the aptamer prior to the signaling of the expression platform. Thus, a complex interplay of several factors has to be taken into account, such as speed of transcription, transcriptional pausing, kinetics and thermodynamics of RNA structure formation, and kinetics and thermodynamics of ligand binding. The response mechanism appears to be best described as a process in which ligand recognition critically dictates the folding pathway of the nascent mRNA during its expression; the resulting structures determine the interactions with the transcriptional or translational apparatus. We discuss experimental methods that offer insight into the dynamics of the free riboswitch state. These include probing experiments, such as in-line and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) techniques, small-angle X-ray scattering (SAXS) analysis, NMR spectroscopy, and fluorescence spectroscopy, including single-molecule fluorescence resonance energy transfer (smFRET) imaging. One of our research contributions is an approach, termed 2ApFold, that incorporates noninvasive 2-aminopurine modifications in riboswitches. The fluorescence response of these moieties is used to delineate the order of secondary–tertiary structure formation and rearrangements taking place during ligand-induced folding. This information can be used to explore the kinetics of ligand recognition and to analyze the degree of structure preorganization of the free riboswitch state. Furthermore, we discuss a recent smFRET study on the SAM-II riboswitch; this report underscores the importance of choosing strategic labeling patterns that leave maximal conformational freedom to the regulatory interaction. Finally, we comment on how riboswitch ligand recognition appeals to the concepts of conformational selection and induced fit, and on the question of whether riboswitches act under thermodynamic or kinetic control. This Account highlights the fact that a thorough understanding of RNA dynamics in vitro is required to shed light on cellular riboswitch mechanisms. Elucidating these mechanisms will contribute not only to ongoing efforts to target riboswitches with antibiotics but also to attempts to engineer artificial cell regulation systems.
A precise tertiary structure must be adopted to allow the function of many RNAs in cells. Accordingly, increasing resources have been devoted to the elucidation of RNA structures and the folding of ...RNAs. 2-Aminopurine (2AP), a fluorescent nucleobase analogue, can be substituted in strategic positions of DNA or RNA molecules to act as site-specific probe to monitor folding and folding dynamics of nucleic acids. Recent studies further demonstrated the potential of 2AP modifications in the assessment of folding kinetics during ligand-induced secondary and tertiary RNA structure rearrangements. However, an efficient way to unambiguously identify reliable positions for 2AP sensors is as yet unavailable and would represent a major asset, especially in the absence of crystallographic or NMR structural data for a target molecule. We report evidence of a novel and direct correlation between the 2′-OH flexibility of nucleotides, observed by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) probing and the fluorescence response following nucleotide substitutions by 2AP. This correlation leads to a straightforward method, using SHAPE probing with benzoyl cyanide, to select appropriate nucleotide sites for 2AP substitution. This clear correlation is presented for three model RNAs of biological significance: the SAM-II, adenine (addA), and preQ1 class II (preQ1cII) riboswitches.
Structural and dynamic features of RNA folding landscapes represent critical aspects of RNA function in the cell and are particularly central to riboswitch-mediated control of gene expression. Here, ...using single-molecule fluorescence energy transfer imaging, we explore the folding dynamics of the preQ ₁ class II riboswitch, an upstream mRNA element that regulates downstream encoded modification enzymes of queuosine biosynthesis. For reasons that are not presently understood, the classical pseudoknot fold of this system harbors an extra stem–loop structure within its 3′-terminal region immediately upstream of the Shine–Dalgarno sequence that contributes to formation of the ligand-bound state. By imaging ligand-dependent preQ ₁ riboswitch folding from multiple structural perspectives, we reveal that the extra stem–loop strongly influences pseudoknot dynamics in a manner that decreases its propensity to spontaneously fold and increases its responsiveness to ligand binding. We conclude that the extra stem–loop sensitizes this RNA to broaden the dynamic range of the ON/OFF regulatory switch.
Post-synthetic modifications of nucleic acids have long been known to affect their functional and structural properties. For instance, numerous different chemical modifications modulate the ...structural organization, stability or translation efficiency of tRNAs and rRNAs. In contrast, little is known about modifications of poly(A)RNAs. Here, we demonstrate for the first time that the two well-studied regulatory long non-coding RNAs HOTAIR and XIST are targets of site-specific cytosine methylation. In both XIST and HOTAIR, we found methylated cytosines located within or near functionally important regions that are known to mediate interaction with chromatin-associated protein complexes. We show that cytosine methylation in the XIST A structure strongly affects binding to the chromatin-modifying complex PRC2 in vitro. These results suggest that cytosine methylation may serve as a general strategy to regulate the function of long non-coding RNAs.
The complex class of cobalamin‐sensitive riboswitches use a “kissing loop” to regulate gene expression. The molecular details of the recognition and folding mode of this riboswitch have been revealed ...by recent X‐ray structures. These insights, together with the identification of RNA polymerase pause sites during transcription, have resulted in a more complete understanding of the response mechanism. AdoCbl=adenosylcobalamin.
Decapping enzymes are required for the removal of the 5'-end m⁷GpppN cap of mRNAs to allow their decay in cells. While many cap-binding proteins recognize the cap structure via the stacking of the ...methylated guanosine ring between two aromatic residues, the precise mechanism of cap recognition by decapping enzymes has yet to be determined. In order to get insights into the interaction of decapping enzymes with the cap structure, we studied the vaccinia virus D10 decapping enzyme as a model to investigate the important features for substrate recognition by the enzyme. We demonstrate that a number of chemically modified purines can competitively inhibit the decapping reaction, highlighting the molecular features of the cap structure that are required for recognition by the enzyme, such as the nature of the moiety at positions 2 and 6 of the guanine base. A 3D structural model of the D10 protein was generated which suggests amino acids implicated in cap binding. Consequently, we expressed 17 mutant proteins with amino acid substitutions in the active site of D10 and found that eight are critical for the decapping activity. These data underscore the functional features involved in the non-canonical cap-recognition by the vaccinia virus D10 decapping enzyme.
Decapping enzymes are required for the removal of the 5'-end cap of mRNAs. These enzymes exhibit a specific hydrolase activity, resulting in cleavage between the alpha- and beta-phosphates of the ...m7GpppN cap to generate both m7GDP and monophosphorylated RNA products. Decapping enzymes have been found in humans, plants and yeasts, and have been discovered more recently in vaccinia virus (D10 protein). Although experimental evidences are lacking, three-metal- and two-metal-ion mechanisms have been proposed so far for the decapping enzymes. In the present study, we performed a biochemical characterization of the interaction of bivalent cations with the vaccinia virus D10 protein. Synergistic activation of the enzyme was observed in the presence of Mg2+ and Mn2+ ions, suggesting the existence of two metal-ion-binding sites on the D10 protein. Moreover, dual-ligand titration experiments using fluorescence spectroscopy demonstrated the presence of two metal-ion-binding sites on the enzyme. A three-dimensional structural model of the active site of the enzyme was generated which highlighted the importance of three glutamate residues involved in the co-ordination of two metal ions and a water molecule. Mutational analyses confirmed the role of two glutamate residues for the binding of metal ions. We demonstrate that one metal ion is co-ordinated by Glu132, while the second metal ion is co-ordinated by Glu145. Taken together, these results support the proposed two-metal-ion mechanistic model for the D10 decapping enzyme.
Most regulatory RNA molecules must adopt a precise secondary fold and tertiary structure to allow their function in cells. A number of experimental approaches, such as the 2-Aminopurine-Based RNA ...Folding Analysis (2ApFold), have therefore been developed to offer insights into the folding and folding dynamics of RNA. A crucial requirement for this method is the selection of proper 2AP labeling positions. In that regard, we recently discovered that Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) offers a reliable path to identify appropriate nucleotides for 2AP substitution on a target RNA. This chapter describes the straightforward procedure to select 2AP substitution sites in RNA molecules using SHAPE probing. The protocols detail the preparation of the target RNA by transcription, and the SHAPE steps including (1) probing of the RNA, (2) reverse transcription with a radiolabeled primer, (3) sequencing gel, and (4) analysis of the obtained band pattern.
The Chlorella virus RNA triphosphatase (cvRTPase) is involved in the formation of the RNA cap structure found at the 5′-end of the viral mRNAs and requires magnesium ions to mediate its catalytic ...activity. To extend our studies on the role of metal ions in phosphohydrolysis, we have used a combination of fluorescence spectroscopy, circular dichroism, denaturation studies and thermodynamic analyses to monitor the binding of magnesium ions to the cvRTPase. Using these techniques, the thermodynamic forces responsible for the interaction of metal ions with an RNA triphosphatase were also evaluated for the first time. Our thermodynamic analyses indicate that the initial association of magnesium with the cvRTPase is dominated by a favorable entropic effect and is accompanied by the release of eight water molecules from the enzyme. Moreover, both fluorescence spectroscopy and circular dichroism assays indicated that minor conformational changes were occurring upon magnesium binding. Mutational studies were also performed and confirmed the importance of three specific glutamate residues located in the active site of the enzyme for the binding of magnesium ions. Finally, in contrast to the yeast RNA triphosphatase, we demonstrate that the binding of magnesium ions to the cvRTPase does not lead to the stabilization of the ground state binding of the RNA substrate. Based on the results of the present study, we hypothesize that the binding of magnesium ions induces local conformational perturbations in the active site residues that ultimately positions the lateral chains of critical amino acids involved in catalysis. Our results highlight fundamental differences in the role of magnesium ions in the phosphohydrolase reactions catalyzed by the cvRTPase and the closely related yeast RNA triphosphatase.
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