RESUMEN
Bacteria live in a broad range of environmental temperatures that require adaptations of their RNA sequences to maintain function. Riboswitches are regulatory RNAs that change conformation upon typically binding metabolite ligands to control bacterial gene expression. The paradigmatic small class-I preQ1 riboswitches from the mesophile Bacillus subtilis (Bsu) and the thermophile Thermoanaerobacter tengcongensis (Tte) adopt similar pseudoknot structures when bound to preQ1. Here, we use UV-melting analysis combined with single-molecule detected chemical denaturation by urea to compare the thermodynamic and kinetic folding properties of the two riboswitches, and the urea-countering effects of trimethylamine N-oxide (TMAO). Our results show that, first, the Tte riboswitch is more thermotolerant than the Bsu riboswitch, despite only subtle sequence differences. Second, using single-molecule FRET, we find that urea destabilizes the folded pseudoknot structure of both riboswitches, yet has a lower impact on the unfolding kinetics of the thermodynamically less stable Bsu riboswitch. Third, our analysis shows that TMAO counteracts urea denaturation and promotes folding of both the riboswitches, albeit with a smaller effect on the more stable Tte riboswitch. Together, these findings elucidate how subtle sequence adaptations in a thermophilic bacterium can stabilize a common RNA structure when a new ecological niche is conquered.
Asunto(s)
Riboswitch , Riboswitch/genética , Transferencia Resonante de Energía de Fluorescencia , Metilaminas , Bacterias/genética , Conformación de Ácido Nucleico , Ligandos , Pliegue del ARNRESUMEN
Late endosome-resident interferon-induced transmembrane protein 3 (IFITM3) inhibits fusion of diverse viruses, including Influenza A virus (IAV), by a poorly understood mechanism. Despite the broad antiviral activity of IFITM3, viruses like Lassa virus (LASV), are fully resistant to its inhibitory effects. It is currently unclear whether resistance arises from a highly efficient fusion machinery that is capable of overcoming IFITM3 restriction or the ability to enter from cellular sites devoid of this factor. Here, we constructed and validated a functional IFITM3 tagged with EGFP or other fluorescent proteins. This breakthrough allowed live cell imaging of virus co-trafficking and fusion with endosomal compartments in cells expressing fluorescent IFITM3. Three-color single virus and endosome tracking revealed that sensitive (IAV), but not resistant (LASV), viruses become trapped within IFITM3-positive endosomes where they underwent hemifusion but failed to release their content into the cytoplasm. IAV fusion with IFITM3-containing compartments could be rescued by amphotericin B treatment, which has been previously shown to antagonize the antiviral activity of this protein. By comparison, virtually all LASV particles trafficked and fused with endosomes lacking detectable levels of fluorescent IFITM3, implying that this virus escapes restriction by utilizing endocytic pathways that are distinct from the IAV entry pathways. The importance of virus uptake and transport pathways is further reinforced by the observation that LASV glycoprotein-mediated cell-cell fusion is inhibited by IFITM3 and other members of the IFITM family expressed in target cells. Together, our results strongly support a model according to which IFITM3 accumulation at the sites of virus fusion is a prerequisite for its antiviral activity and that this protein traps viral fusion at a hemifusion stage by preventing the formation of fusion pores. We conclude that the ability to utilize alternative endocytic pathways for entry confers IFITM3-resistance to otherwise sensitive viruses.
Asunto(s)
Endosomas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/fisiología , Células A549/metabolismo , Animales , Antivirales/metabolismo , Células COS/metabolismo , Chlorocebus aethiops , Endosomas/virología , Células HEK293/metabolismo , Interacciones Huésped-Patógeno , Humanos , Virus de la Influenza A/patogenicidad , Interferones/metabolismo , Virus Lassa/patogenicidad , Imagen Óptica/métodos , Transporte de Proteínas , Internalización del VirusRESUMEN
T-box riboswitches are a widespread class of structured noncoding RNAs in Gram-positive bacteria that regulate the expression of amino acid-related genes. They form negative feedback loops to maintain steady supplies of aminoacyl-transfer RNAs (tRNAs) to the translating ribosomes. T-box riboswitches are located in the 5' leader regions of mRNAs that they regulate and directly bind to their cognate tRNA ligands. T-boxes further sense the aminoacylation state of the bound tRNAs and, based on this readout, regulate gene expression at the level of transcription or translation. T-box riboswitches consist of two conserved domains-a 5' Stem I domain that is involved in specific tRNA recognition and a 3' antiterminator/antisequestrator (or discriminator) domain that senses the amino acid on the 3' end of the bound tRNA. Interaction of the 3' end of an uncharged but not charged tRNA with a thermodynamically weak discriminator domain stabilizes it to promote transcription readthrough or translation initiation. Recent biochemical, biophysical, and structural studies have provided high-resolution insights into the mechanism of tRNA recognition by Stem I, several structural models of full-length T-box-tRNA complexes, mechanism of amino acid sensing by the antiterminator domain, as well as kinetic details of tRNA binding to the T-box riboswitches. In addition, translation-regulating T-box riboswitches have been recently characterized, which presented key differences from the canonical transcriptional T-boxes. Here, we review the recent developments in understanding the T-box riboswitch mechanism that have employed various complementary approaches. Further, the regulation of multiple essential genes by T-boxes makes them very attractive drug targets to combat drug resistance. The recent progress in understanding the biochemical, structural, and dynamic aspects of the T-box riboswitch mechanism will enable more precise and effective targeting with small molecules. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1167-1180, 2019.
Asunto(s)
Conformación de Ácido Nucleico , ARN/química , Riboswitch , Antibacterianos , Bacillus subtilis/metabolismo , Sitios de Unión , Codón , Ligandos , Biosíntesis de Proteínas , Dominios Proteicos , Pliegue de Proteína , ARN Bacteriano/química , ARN de Transferencia/química , ARN de Transferencia de Tirosina/química , Termodinámica , Transcripción Genética , Tirosina-ARNt Ligasa/genéticaRESUMEN
PreQ1-III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HLout-type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ1-III riboswitch aptamer forms a HLout-type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ1 binds tightly (KD,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3' RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ1 enhances P4 reorientation toward P1-P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (kdock â¼ 0.6 s(-1)) and undocking (kundock â¼ 1.1 s(-1)). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs.
Asunto(s)
Aptámeros de Nucleótidos/genética , ARN Bacteriano/genética , Ribosomas/genética , Riboswitch/genética , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Clostridium/genética , Clostridium/metabolismo , Cristalografía por Rayos X , Cinética , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Nucleósido Q/química , Nucleósido Q/metabolismo , Pirimidinonas/química , Pirimidinonas/metabolismo , Pirroles/química , Pirroles/metabolismo , Pliegue del ARN , ARN Bacteriano/química , ARN Bacteriano/metabolismo , Ribosomas/metabolismo , TermodinámicaRESUMEN
Bacterial riboswitches couple small-molecule ligand binding to RNA conformational changes that widely regulate gene expression, rendering them potential targets for antibiotic intervention. Despite structural insights, the ligand-mediated folding mechanisms of riboswitches are still poorly understood. Using single-molecule fluorescence resonance energy transfer (smFRET), we have investigated the folding mechanism of an H-type pseudoknotted preQ1 riboswitch in dependence of Mg(2+) and three ligands of distinct affinities. We show that, in the absence of Mg(2+), both weakly and strongly bound ligands promote pseudoknot docking through an induced-fit mechanism. By contrast, addition of as low as 10 µM Mg(2+) generally shifts docking toward conformational selection by stabilizing a folded-like conformation prior to ligand binding. Supporting evidence from transition-state analysis further highlights the particular importance of stacking interactions during induced-fit and of specific hydrogen bonds during conformational selection. Our mechanistic dissection provides unprecedented insights into the intricate synergy between ligand- and Mg(2+)-mediated RNA folding.
Asunto(s)
Magnesio/farmacología , Conformación de Ácido Nucleico/efectos de los fármacos , Pliegue del ARN/efectos de los fármacos , ARN Bacteriano/química , Riboswitch , Transferencia Resonante de Energía de Fluorescencia , Enlace de Hidrógeno , Ligandos , Magnesio/químicaRESUMEN
Four days after the announcement of the 2014 Nobel Prize in Chemistry for "the development of super-resolved fluorescence microscopy" based on single molecule detection, the Single Molecule Analysis in Real-Time (SMART) Center at the University of Michigan hosted a "Principles of Single Molecule Techniques 2014" course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines.
Asunto(s)
Microscopía Fluorescente , Congresos como AsuntoRESUMEN
Riboswitches are structural elements in the 5' untranslated regions of many bacterial messenger RNAs that regulate gene expression in response to changing metabolite concentrations by inhibition of either transcription or translation initiation. The preQ1 (7-aminomethyl-7-deazaguanine) riboswitch family comprises some of the smallest metabolite sensing RNAs found in nature. Once ligand-bound, the transcriptional Bacillus subtilis and translational Thermoanaerobacter tengcongensis preQ1 riboswitch aptamers are structurally similar RNA pseudoknots; yet, prior structural studies have characterized their ligand-free conformations as largely unfolded and folded, respectively. In contrast, through single molecule observation, we now show that, at near-physiological Mg(2+) concentration and pH, both ligand-free aptamers adopt similar pre-folded state ensembles that differ in their ligand-mediated folding. Structure-based Go-model simulations of the two aptamers suggest that the ligand binds late (Bacillus subtilis) and early (Thermoanaerobacter tengcongensis) relative to pseudoknot folding, leading to the proposal that the principal distinction between the two riboswitches lies in their relative tendencies to fold via mechanisms of conformational selection and induced fit, respectively. These mechanistic insights are put to the test by rationally designing a single nucleotide swap distal from the ligand binding pocket that we find to predictably control the aptamers' pre-folded states and their ligand binding affinities.
Asunto(s)
Biosíntesis de Proteínas , Pirimidinonas/metabolismo , Pirroles/metabolismo , Riboswitch , Transcripción Genética , Bacillus subtilis/genética , Transferencia Resonante de Energía de Fluorescencia , Ligandos , Conformación de Ácido Nucleico , Pliegue del ARN , Thermoanaerobacter/genéticaRESUMEN
Single-stranded RNAs (ssRNAs) are ubiquitous RNA elements that serve diverse functional roles. Much of our understanding of ssRNA conformational behavior is limited to structures in which ssRNA directly engages in tertiary interactions or is recognized by proteins. Little is known about the structural and dynamic behavior of free ssRNAs at atomic resolution. Here, we report the collaborative application of nuclear magnetic resonance (NMR) and replica exchange molecular dynamics (REMD) simulations to characterize the 12 nt ssRNA tail derived from the prequeuosine riboswitch. NMR carbon spin relaxation data and residual dipolar coupling measurements reveal a flexible yet stacked core adopting an A-form-like conformation, with the level of order decreasing toward the terminal ends. An A-to-C mutation within the polyadenine tract alters the observed dynamics consistent with the introduction of a dynamic kink. Pre-ordering of the tail may increase the efficacy of ligand binding above that achieved by a random-coil ssRNA. The REMD simulations recapitulate important trends in the NMR data, but suggest more internal motions than inferred from the NMR analysis. Our study unmasks a previously unappreciated level of complexity in ssRNA, which we believe will also serve as an excellent model system for testing and developing computational force fields.
Asunto(s)
Pirimidinonas/química , Pirroles/química , ARN/química , Riboswitch , Adenina/química , Ligandos , Simulación de Dinámica Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Estabilidad del ARNRESUMEN
T-box riboswitches are multi-domain noncoding RNAs that surveil individual amino acid availabilities in most Gram-positive bacteria. T-boxes directly bind specific tRNAs, query their aminoacylation status to detect starvation, and feedback control the transcription or translation of downstream amino-acid metabolic genes. Most T-boxes rapidly recruit their cognate tRNA ligands through an intricate three-way stem I-stem II-tRNA interaction, whose establishment is not understood. Using single-molecule FRET, SAXS, and time-resolved fluorescence, we find that the free T-box RNA assumes a broad distribution of open, semi-open, and closed conformations that only slowly interconvert. tRNA directly binds all three conformers with distinct kinetics, triggers nearly instantaneous collapses of the open conformations, and returns the T-box RNA to their pre-binding conformations upon dissociation. This scissors-like dynamic behavior is enabled by a hinge-like pseudoknot domain which poises the T-box for rapid tRNA-induced domain closure. This study reveals tRNA-chaperoned folding of flexible, multi-domain mRNAs through a Venus flytrap-like mechanism.
Asunto(s)
Pliegue del ARN , Riboswitch , Dispersión del Ángulo Pequeño , Difracción de Rayos X , ARN , Riboswitch/genética , Aminoácidos , Chaperonas MolecularesRESUMEN
T-box riboswitches are modular bacterial noncoding RNAs that sense and regulate amino acid availability through direct interactions with tRNAs. Between the 5' anticodon-binding stem I domain and the 3' amino acid sensing domains of most T-boxes lies the stem II domain of unknown structure and function. Here, we report a 2.8-Å cocrystal structure of the Nocardia farcinica ileS T-box in complex with its cognate tRNAIle. The structure reveals a perpendicularly arranged ultrashort stem I containing a K-turn and an elongated stem II bearing an S-turn. Both stems rest against a compact pseudoknot, dock via an extended ribose zipper and jointly create a binding groove specific to the anticodon of its cognate tRNA. Contrary to proposed distal contacts to the tRNA elbow region, stem II locally reinforces the codon-anticodon interactions between stem I and tRNA, achieving low-nanomolar affinity. This study illustrates how mRNA junctions can create specific binding sites for interacting RNAs of prescribed sequence and structure.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Isoleucina-ARNt Ligasa/genética , Nocardia/genética , Motivos de Nucleótidos , ARN Bacteriano/química , ARN de Transferencia de Isoleucina/química , Riboswitch/genética , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , ARN Bacteriano/metabolismo , ARN de Transferencia de Isoleucina/metabolismo , Relación Estructura-ActividadRESUMEN
The widespread Mn2+-sensing yybP-ykoY riboswitch controls the expression of bacterial Mn2+ homeostasis genes. Here, we first determine the crystal structure of the ligand-bound yybP-ykoY riboswitch aptamer from Xanthomonas oryzae at 2.96 Å resolution, revealing two conformations with docked four-way junction (4WJ) and incompletely coordinated metal ions. In >100 µs of MD simulations, we observe that loss of divalents from the core triggers local structural perturbations in the adjacent docking interface, laying the foundation for signal transduction to the regulatory switch helix. Using single-molecule FRET, we unveil a previously unobserved extended 4WJ conformation that samples transient docked states in the presence of Mg2+. Only upon adding sub-millimolar Mn2+, however, can the 4WJ dock stably, a feature lost upon mutation of an adenosine contacting Mn2+ in the core. These observations illuminate how subtly differing ligand preferences of competing metal ions become amplified by the coupling of local with global RNA dynamics.
Asunto(s)
Magnesio/metabolismo , ARN Bacteriano/química , ARN Bacteriano/metabolismo , Riboswitch/fisiología , Transducción de Señal , Xanthomonas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Ligandos , Manganeso/metabolismo , Modelos Moleculares , Conformación Molecular , Simulación de Dinámica Molecular , Mutación , Conformación de Ácido Nucleico , ARN Bacteriano/genéticaRESUMEN
In Gram-positive bacteria, T-box riboswitches control gene expression to maintain the cellular pools of aminoacylated tRNAs essential for protein biosynthesis. Co-transcriptional binding of an uncharged tRNA to the riboswitch stabilizes an antiterminator, allowing transcription read-through, whereas an aminoacylated tRNA does not. Recent structural studies have resolved two contact points between tRNA and Stem-I in the 5' half of the T-box riboswitch, but little is known about the mechanism empowering transcriptional control by a small, distal aminoacyl modification. Using single-molecule fluorescence microscopy, we have probed the kinetic and structural underpinnings of tRNA binding to a glycyl T-box riboswitch. We observe a two-step mechanism where fast, dynamic recruitment of tRNA by Stem-I is followed by ultra-stable anchoring by the downstream antiterminator, but only without aminoacylation. Our results support a hierarchical sensing mechanism wherein dynamic global binding of the tRNA body is followed by localized readout of its aminoacylation status by snap-lock-based trapping.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Bacterias Grampositivas/genética , ARN Bacteriano/química , ARN de Transferencia/química , Riboswitch , Emparejamiento Base , Bacterias Grampositivas/metabolismo , Microscopía Fluorescente , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Imagen Individual de Molécula , Aminoacilación de ARN de TransferenciaRESUMEN
The recent discovery that non-coding RNAs are considerably more abundant and serve a much wider range of critical cellular functions than recognized over previous decades of research into molecular biology has sparked a renewed interest in the study of structure-function relationships of RNA. To perform their functions in the cell, RNAs must dominantly adopt their native conformations, avoiding deep, non-productive kinetic traps that may exist along a frustrated (rugged) folding free energy landscape. Intracellularly, RNAs are synthesized by RNA polymerase and fold co-transcriptionally starting from the 5' end, sometimes with the aid of protein chaperones. By contrast, in the laboratory RNAs are commonly generated by in vitro transcription or chemical synthesis, followed by purification in a manner that includes the use of high concentrations of urea, heat and UV light (for detection), resulting in the denaturation and subsequent refolding of the entire RNA. Recent studies into the nature of heterogeneous RNA populations resulting from this process have underscored the need for non-denaturing (native) purification methods that maintain the co-transcriptional fold of an RNA. Here, we present protocols for the native purification of an RNA after its in vitro transcription and for fluorophore and biotin labeling methods designed to preserve its native conformation for use in single molecule fluorescence resonance energy transfer (smFRET) inquiries into its structure and function. Finally, we present methods for taking smFRET data and for analyzing them, as well as a description of plausible overall preparation schemes for the plethora of non-coding RNAs.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , ARN/aislamiento & purificación , Coloración y Etiquetado , Animales , Bencenosulfonatos , Biotinilación , Bovinos , Química Clic , ARN Polimerasas Dirigidas por ADN/metabolismo , Colorantes Fluorescentes/metabolismo , Oligonucleótidos/metabolismo , Ácido Peryódico/química , Poli A/metabolismo , Polietilenglicoles/química , ARN/metabolismo , Riboswitch , Albúmina Sérica Bovina/metabolismo , Estreptavidina/metabolismo , Thermoanaerobacter/metabolismo , Transcripción Genética , Proteínas Virales/metabolismoRESUMEN
Riboswitches are structured noncoding RNA elements that control the expression of their embedding messenger RNAs by sensing the intracellular concentration of diverse metabolites. As the name suggests, riboswitches are dynamic in nature so that studying their inherent conformational dynamics and ligand-mediated folding is important for understanding their mechanism of action. Single-molecule fluorescence energy transfer (smFRET) microscopy is a powerful and versatile technique for studying the folding pathways and intra- and intermolecular dynamics of biological macromolecules, especially RNA. The ability of smFRET to monitor intramolecular distances and their temporal evolution make it a particularly insightful tool for probing the structure and dynamics of riboswitches. Here, we detail the general steps for using prism-based total internal reflection fluorescence microscopy for smFRET studies of the structure, dynamics, and ligand-binding mechanisms of riboswitches.
Asunto(s)
Microscopía Fluorescente/métodos , Riboswitch , Diseño de Equipo , Microscopía Fluorescente/instrumentación , Conformación de Ácido Nucleico , Pliegue del ARNRESUMEN
Capsules frequently play a key role in bacterial interactions with their environment. Escherichia coli capsules were categorized as groups 1 through 4, each produced by a distinct mechanism. Etk and Etp are members of protein families required for the production of group 1 and group 4 capsules. These members function as a protein tyrosine kinase and protein tyrosine phosphatase, respectively. We show that Etp dephosphorylates Etk in vivo, and mutations rendering Etk or Etp catalytically inactive result in loss of group 4 capsule production, supporting the notion that cyclic phosphorylation and dephosphorylation of Etk is required for capsule formation. Notably, Etp also becomes tyrosine phosphorylated in vivo and catalyzes rapid auto-dephosphorylation. Further analysis identified Tyr121 as the phosphorylated residue of Etp. Etp containing Phe, Glu or Ala in place of Tyr121 retained phosphatase activity and catalyzed dephosphorylation of Etp and Etk. Although EtpY121E and EtpY121A still supported capsule formation, EtpY121F failed to do so. These results suggest that cycles of phosphorylation and dephosphorylation of Etp, as well as Etk, are involved in the formation of group 4 capsule, providing an additional regulatory layer to the complex control of capsule production.
Asunto(s)
Cápsulas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Proteínas de la Membrana/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Secuencia de Aminoácidos , Cinética , Modelos Biológicos , Datos de Secuencia Molecular , Mutación/genética , Fosforilación , Fosfotirosina/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The enzyme phospholipase C-ß (PLCß) is a crucial regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to members of the Gq family of heterotrimeric G proteins. We have determined atomic structures of two invertebrate homologs of PLCß (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLCß3 considerably increase basal activity and diminish stimulation by Gαq. Gαq binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLCß.