RESUMEN
The origin of life likely occurred within environments that concentrated cellular precursors and enabled their co-assembly into cells. Soda lakes (those dominated by Na+ ions and carbonate species) can concentrate precursors of RNA and membranes, such as phosphate, cyanide, and fatty acids. Subsequent assembly of RNA and membranes into cells is a long-standing problem because RNA function requires divalent cations, e.g. Mg2+, but Mg2+ disrupts fatty acid membranes. The low solubility of Mg-containing carbonates limits soda lakes to moderate Mg2+ concentrations (â¼1â mM), so we investigated whether both RNAs and membranes function within these lakes. We collected water from Last Chance Lake and Goodenough Lake in Canada. Because we sampled after seasonal evaporation, the lake water contained â¼1â M Na+ and â¼1â mMâ Mg2+ near pH 10. In the laboratory, nonenzymatic, RNA-templated polymerization of 2-aminoimidazole-activated ribonucleotides occurred at comparable rates in lake water and standard laboratory conditions (50â mM MgCl2, pH 8). Additionally, we found that a ligase ribozyme that uses oligonucleotide substrates activated with 2-aminoimidazole was active in lake water after adjusting pH from â¼10 to 9. We also observed that decanoic acid and decanol assembled into vesicles in a dilute solution that resembled lake water after seasonal rains, and that those vesicles retained encapsulated solutes despite salt-induced flocculation when the external solution was replaced with dry-season lake water. By identifying compatible conditions for nonenzymatic and ribozyme-catalyzed RNA assembly, and for encapsulation by membranes, our results suggest that soda lakes could have enabled cellular life to emerge on Earth, and perhaps elsewhere.
RESUMEN
Catalytic RNAs or ribozymes are considered to be central to primordial biology. Most ribozymes require moderate to high concentrations of divalent cations such as Mg2+ to fold into their catalytically competent structures and perform catalysis. However, undesirable effects of Mg2+ such as hydrolysis of reactive RNA building blocks and degradation of RNA structures are likely to undermine its beneficial roles in ribozyme catalysis. Further, prebiotic cell-like compartments bounded by fatty acid membranes are destabilized in the presence of Mg2+, making ribozyme function inside prebiotically relevant protocells a significant challenge. Therefore, we sought to identify conditions that would enable ribozymes to retain activity at low concentrations of Mg2+. Inspired by the ability of ribozymes to function inside crowded cellular environments with <1 mM free Mg2+, we tested molecular crowding as a potential mechanism to lower the Mg2+ concentration required for ribozyme-catalyzed RNA assembly. Here, we show that the ribozyme-catalyzed ligation of phosphorimidazolide RNA substrates is significantly enhanced in the presence of the artificial crowding agent polyethylene glycol. We also found that molecular crowding preserves ligase activity under denaturing conditions such as alkaline pH and the presence of urea. Additionally, we show that crowding-induced stimulation of RNA-catalyzed RNA assembly is not limited to phosphorimidazolide ligation but extends to the RNA-catalyzed polymerization of nucleoside triphosphates. RNA-catalyzed RNA ligation is also stimulated by the presence of prebiotically relevant small molecules such as ethylene glycol, ribose, and amino acids, consistent with a role for molecular crowding in primordial ribozyme function and more generally in the emergence of RNA-based cellular life.
RESUMEN
Demonstrating RNA catalysis within prebiotically relevant models of primordial cells (protocells) remains a challenge in origins of life research. Fatty acid vesicles encapsulating genomic and catalytic RNAs (ribozymes) are attractive models for protocells; however, RNA catalysis has largely been incompatible with fatty acid vesicles due to their instability in the presence of Mg2+ at the concentrations required for ribozyme function. Here, we report a ribozyme that catalyzes template-directed RNA ligation at low Mg2+ concentrations and thus remains active within stable vesicles. Ribose and adenine, both prebiotically relevant molecules, were found to greatly reduce Mg2+ -induced RNA leakage from vesicles. When we co-encapsulated the ribozyme, substrate, and template within fatty acid vesicles, we observed efficient RNA-catalyzed RNA ligation upon subsequent addition of Mg2+ . Our work shows that RNA-catalyzed RNA assembly can occur efficiently within prebiotically plausible fatty acid vesicles and represents a step toward the replication of primordial genomes within self-replicating protocells.
Asunto(s)
Células Artificiales , ARN Catalítico , ARN/química , ARN Catalítico/química , Ácidos Grasos , CatálisisRESUMEN
The Prebiotic Chemistry and Early Earth Environments (PCE3) Consortium is a community of researchers seeking to understand the origins of life on Earth and in the universe. PCE3 is one of five Research Coordination Networks (RCNs) within NASA's Astrobiology Program. Here we report on the inaugural PCE3 workshop, intended to cross-pollinate, transfer information, promote cooperation, break down disciplinary barriers, identify new directions, and foster collaborations. This workshop, entitled, "Building a New Foundation", was designed to propagate current knowledge, identify possibilities for multidisciplinary collaboration, and ultimately define paths for future collaborations. Presentations addressed the likely conditions on early Earth in ways that could be incorporated into prebiotic chemistry experiments and conceptual models to improve their plausibility and accuracy. Additionally, the discussions that followed among workshop participants helped to identify within each subdiscipline particularly impactful new research directions. At its core, the foundational knowledge base presented in this workshop should underpin future workshops and enable collaborations that bridge the many disciplines that are part of PCE3.
Asunto(s)
Planeta Tierra , Origen de la Vida , Humanos , Modelos TeóricosRESUMEN
Next-generation sequencing (NGS) enables the identification of functional nucleic acid sequences from in vitro selection/evolution experiments and illuminates the evolutionary process at single-nucleotide resolution. However, analyzing the vast output from NGS can be daunting, especially with limited programming skills. We developed REVERSE (Rapid EValuation of Experimental RNA Selection/Evolution) (https://www.reverseserver.org/), a web server that implements an integrated computational pipeline through a graphical user interface, which performs both pre-processing and detailed sequence level analyses within minutes. Raw FASTQ files are quality-filtered, dereplicated, and trimmed before being analyzed by either of two pipelines. The first pipeline counts, sorts, and tracks enrichment of unique sequences and user-defined sequence motifs. It also identifies mutational intermediates present in the sequence data that connect two input sequences. The second pipeline sorts similar sequences into clusters and tracks enrichment of peak sequences. It also performs nucleotide conservation analysis on the cluster of choice and generates a consensus sequence. Both pipelines generate downloadable spreadsheets and high-resolution figures. Collectively, REVERSE is a one-stop solution for the rapid analysis of NGS data obtained from in vitro selection/evolution experiments that obviates the need for computational expertise.
Asunto(s)
Evolución Molecular Dirigida , Análisis de Secuencia de ARN , Programas Informáticos , Biología Computacional , Computadores , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación , Interfaz Usuario-ComputadorRESUMEN
Aminoacylated transfer RNAs, which harbor a covalent linkage between amino acids and RNA, are a universally conserved feature of life. Because they are essential substrates for ribosomal translation, aminoacylated oligonucleotides must have been present in the RNA world prior to the evolution of the ribosome. One possibility we are exploring is that the aminoacyl ester linkage served another function before being recruited for ribosomal protein synthesis. The nonenzymatic assembly of ribozymes from short RNA oligomers under realistic conditions remains a key challenge in demonstrating a plausible pathway from prebiotic chemistry to the RNA world. Here, we show that aminoacylated RNAs can undergo template-directed assembly into chimeric amino acid-RNA polymers that are active ribozymes. We demonstrate that such chimeric polymers can retain the enzymatic function of their all-RNA counterparts by generating chimeric hammerhead, RNA ligase, and aminoacyl transferase ribozymes. Amino acids with diverse side chains form linkages that are well tolerated within the RNA backbone and, in the case of an aminoacyl transferase, even in its catalytic center, potentially bringing novel functionalities to ribozyme catalysis. Our work suggests that aminoacylation chemistry may have played a role in primordial ribozyme assembly. Increasing the efficiency of this process provides an evolutionary rationale for the emergence of sequence and amino acid-specific aminoacyl-RNA synthetase ribozymes, which could then have generated the substrates for ribosomal protein synthesis.
Asunto(s)
ARN Catalítico/metabolismo , Aminoacilación de ARN de Transferencia/fisiología , Secuencia de Bases , ADN , Conformación de Ácido Nucleico , ARN de Transferencia/genética , ARN de Transferencia/metabolismoRESUMEN
Ribozymes that react with small-molecule probes have important applications in transcriptomics and chemical biology, such as RNA labeling and imaging. Understanding the structural basis for these RNA-modifying reactions will enable the development of better tools for studying RNA. Nevertheless, high-resolution structures and underlying catalytic mechanisms for members of this ribozyme class remain elusive. Here, we focus on a self-alkylating ribozyme that catalyzes nitrogen-carbon bond formation between a specific guanine and a 2,3-disubstituted epoxide substrate and report the crystal structures of a self-alkylating ribozyme, including both alkylated and apo forms, at 1.71-Å and 2.49-Å resolution, respectively. The ribozyme assumes an elongated hairpin-like architecture preorganized to accommodate the epoxide substrate in a hook-shaped conformation. Observed reactivity of substrate analogs together with an inverse, log-linear pH dependence of the reaction rate suggests a requirement for epoxide protonation, possibly assisted by the ether oxygens within the substrate.
Asunto(s)
ARN Catalítico , Catálisis , Compuestos Epoxi , Conformación de Ácido Nucleico , ARN , ARN Catalítico/metabolismoRESUMEN
Connections between distinct catalytic RNA motifs through networks of mutations that retain catalytic function (neutral networks) were likely central to the evolution of biocatalysis. Despite suggestions that functional RNAs collectively form an interconnected web of neutral networks, little evidence has emerged to demonstrate the existence of such intersecting networks in naturally occurring RNAs. Here we show that neutral networks of two naturally occurring, seemingly unrelated endonucleolytic ribozymes, the hammerhead (HH) and hairpin (HP), intersect. Sequences at the intersection of these networks exhibit catalytic functions corresponding to both ribozymes by potentially populating both catalytic folds and enable a smooth crossover between the two. Small and structurally simple endonucleolytic motifs like the HH ribozyme could, through mutational walks along their neutral networks, encounter novel catalytic phenotypes, and structurally flexible, bifunctional sequences at the intersection of these networks could have acted as nodes for evolutionary diversification in an RNA world. Considering the simplicity and small size of the HH ribozyme, we propose that this self-cleaving motif could have been a precursor to other more complex endonucleolytic ribozymes. More generally, our results suggest that RNAs that possess distinct sequences, structures, and catalytic functions, can potentially share evolutionary history through mutational connections in sequence space.
Asunto(s)
ADN/genética , Secuencias Invertidas Repetidas , ARN Catalítico/metabolismo , Transcripción Genética , Emparejamiento Base , Biocatálisis , Sistema Libre de Células , ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Conformación de Ácido Nucleico , Motivos de Nucleótidos , Radioisótopos de Fósforo , Mutación Puntual , ARN Catalítico/química , ARN Catalítico/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The discovery of catalytic RNAs or ribozymes introduced a new class of enzymes to biology. In addition to their increasingly important roles in modern life, ribozymes are key players in the RNA World hypothesis, which posits that life started or flourished with RNA supporting both genetic and enzymatic functions. Therefore, investigations into the mechanisms of ribozyme function provide an exciting opportunity to examine the foundational principles of biological catalysis. Ribozymes are also attractive model systems to investigate the relationship between structure and function in RNA. Endonucleolytic ribozymes represent the largest class of catalytic RNA, of which the Varkud satellite (VS) ribozyme is structurally the most complex. The last ribozyme to be discovered by accident, the VS ribozyme had eluded structural determination for over two decades. When we solved the first crystal structures of the VS ribozyme, an extensive body of biochemical and biophysical data had accumulated over the years with which we could evaluate the functional relevance of the structure. Conversely, the structures provided a new perspective from which to reexamine the functional data and test new hypotheses. The VS ribozyme is organized in a modular fashion where independently folding domains assemble into the active conformation of the ribozyme via three-way junctions. Structures of the VS ribozyme in complex with its substrate at different stages of activation enabled us to map the structural reorganization of the substrate that must precede catalysis. In addition to defining the global architecture of the RNA, the essential interactions between the substrate and catalytic domains, and the rearrangements in the substrate prior to catalysis, these structures provided detailed snapshots of the ribozyme active site, revealing potential catalytic interactions. High resolution structures of the active site bolstered the view that the catalytic mechanism involved nucleobase-mediated general acid-base catalysis and uncovered additional catalytic interactions between the cleavage site and catalytic residues. Informed by the crystal structures of the VS ribozyme, an integrated experimental and computational approach identified the key players and essential interactions that define the active site of the ribozyme. This confluence of biochemical, structural, and computational studies revealed the catalytic mechanism of the ribozyme at unprecedented detail. Additionally, comparative analyses of the active site structures of the VS ribozyme and other nucleic acid-based endoribonucleases revealed common architectural motifs and strikingly similar catalytic strategies. In this Account, we document the progress of VS ribozyme research starting from its discovery and extending to the elucidation of its detailed catalytic mechanism 30 years later.
Asunto(s)
ARN Catalítico , Biocatálisis , Cristalografía por Rayos X , Simulación del Acoplamiento Molecular , ARN Catalítico/química , ARN Catalítico/genética , ARN Catalítico/metabolismoRESUMEN
RNA enzymes or ribozymes catalyze some of the most important reactions in biology and are thought to have played a central role in the origin and evolution of life on earth. Catalytic function in RNA has evolved in crowded cellular environments that are different from dilute solutions in which most in vitro assays are performed. The presence of molecules such as amino acids, polypeptides, alcohols, and sugars in the cell introduces forces that modify the kinetics and thermodynamics of ribozyme-catalyzed reactions. Synthetic molecules are routinely used in in vitro studies to better approximate the properties of biomolecules under in vivo conditions. This review discusses the various forces that operate within simulated crowded solutions in the context of RNA structure, folding, and catalysis. It also explores ideas about how crowding could have been beneficial to the evolution of functional RNAs and the development of primitive cellular systems in a prebiotic milieu.
Asunto(s)
ARNRESUMEN
The hypothesized central role of RNA in the origin of life suggests that RNA propagation predated the advent of complex protein enzymes. A critical step of RNA replication is the template-directed synthesis of a complementary strand. Two experimental approaches have been extensively explored in the pursuit of demonstrating protein-free RNA synthesis: template-directed nonenzymatic RNA polymerization using intrinsically reactive monomers and ribozyme-catalyzed polymerization using more stable substrates such as biological 5'-triphosphates. Despite significant progress in both approaches in recent years, the assembly and copying of functional RNA sequences under prebiotic conditions remains a challenge. Here, we explore an alternative approach to RNA-templated RNA copying that combines ribozyme catalysis with RNA substrates activated with a prebiotically plausible leaving group, 2-aminoimidazole (2AI). We applied in vitro selection to identify ligase ribozymes that catalyze phosphodiester bond formation between a template-bound primer and a phosphor-imidazolide-activated oligomer. Sequencing revealed the progressive enrichment of 10 abundant sequences from a random sequence pool. Ligase activity was detected in all 10 RNA sequences; all required activation of the ligator with 2AI and generated a 3'-5' phosphodiester bond. We propose that ribozyme catalysis of phosphodiester bond formation using intrinsically reactive RNA substrates, such as imidazolides, could have been an evolutionary step connecting purely nonenzymatic to ribozyme-catalyzed RNA template copying during the origin of life.
Asunto(s)
Imidazoles/química , Origen de la Vida , ARN Ligasa (ATP)/química , ARN Catalítico/química , Imidazoles/metabolismo , Polimerizacion , ARN Ligasa (ATP)/metabolismo , ARN Catalítico/metabolismoRESUMEN
We report DNA- and RNA-like systems built from eight nucleotide "letters" (hence the name "hachimoji") that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos.
Asunto(s)
Emparejamiento Base , ADN/química , ADN/genética , Nucleótidos/química , ARN/química , ARN/genética , Cristalografía , Fluorescencia , Conformación de Ácido Nucleico , Polielectrolitos/química , Biología Sintética , TermodinámicaRESUMEN
Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3-6 binds to an AAACA RNA pentaloop closed by a GC pair with â¼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5'-GNGACCC-3' consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab-RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography.
Asunto(s)
Cristalografía por Rayos X , Fragmentos Fab de Inmunoglobulinas/química , ARN/química , ARN/inmunología , Regiones Determinantes de Complementariedad/química , Epítopos/química , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Modelos Moleculares , Mutación , Motivos de NucleótidosRESUMEN
The Varkud satellite (VS) ribozyme catalyzes site-specific RNA cleavage and ligation reactions. Recognition of the substrate involves a kissing loop interaction between the substrate and the catalytic domain of the ribozyme, resulting in a rearrangement of the substrate helix register into a so-called "shifted" conformation that is critical for substrate binding and activation. We report a 3.3 Å crystal structure of the complete ribozyme that reveals the active, shifted conformation of the substrate, docked into the catalytic domain of the ribozyme. Comparison to previous NMR structures of isolated, inactive substrates provides a physical description of substrate remodeling, and implicates roles for tertiary interactions in catalytic activation of the cleavage loop. Similarities to the hairpin ribozyme cleavage loop activation suggest general strategies to enhance fidelity in RNA folding and ribozyme cleavage.
Asunto(s)
Endorribonucleasas/química , ARN Catalítico/química , Biocatálisis , Endorribonucleasas/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , ARN/química , Pliegue del ARN , ARN Catalítico/metabolismo , Especificidad por SustratoRESUMEN
Reported here is a laboratory in vitro evolution (LIVE) experiment based on an artificially expanded genetic information system (AEGIS). This experiment delivers the first example of an AEGIS aptamer that binds to an isolated protein target, the first whose structural contact with its target has been outlined and the first to inhibit biologically important activities of its target, the protective antigen from Bacillus anthracis We show how rational design based on secondary structure predictions can also direct the use of AEGIS to improve the stability and binding of the aptamer to its target. The final aptamer has a dissociation constant of â¼35 nM. These results illustrate the value of AEGIS-LIVE for those seeking to obtain receptors and ligands without the complexities of medicinal chemistry, and also challenge the biophysical community to develop new tools to analyze the spectroscopic signatures of new DNA folds that will emerge in synthetic genetic systems replacing standard DNA and RNA as platforms for LIVE.
Asunto(s)
Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Técnica SELEX de Producción de Aptámeros , Antígenos Bacterianos/inmunología , Bacillus anthracis/genética , Bacillus anthracis/inmunología , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/inmunología , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Dicroismo Circular , G-Cuádruplex , Cinética , Mutación , Conformación de Ácido Nucleico , Relación Estructura-Actividad , Biología SintéticaRESUMEN
The Varkud satellite (VS) ribozyme mediates rolling-circle replication of a plasmid found in the Neurospora mitochondrion. We report crystal structures of this ribozyme from Neurospora intermedia at 3.1 Å resolution, which revealed an intertwined dimer formed by an exchange of substrate helices. In each protomer, an arrangement of three-way helical junctions organizes seven helices into a global fold that creates a docking site for the substrate helix of the other protomer, resulting in the formation of two active sites in trans. This mode of RNA-RNA association resembles the process of domain swapping in proteins and has implications for RNA regulation and evolution. Within each active site, adenine and guanine nucleobases abut the scissile phosphate, poised to serve direct roles in catalysis. Similarities to the active sites of the hairpin and hammerhead ribozymes highlight the functional importance of active-site features, underscore the ability of RNA to access functional architectures from distant regions of sequence space, and suggest convergent evolution.
Asunto(s)
Endorribonucleasas/química , Proteínas Fúngicas/química , Neurospora/química , ARN Catalítico/química , ARN/química , Adenina/química , Adenina/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Evolución Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expresión Génica , Guanina/química , Guanina/metabolismo , Mitocondrias/química , Mitocondrias/enzimología , Simulación del Acoplamiento Molecular , Mutación , Neurospora/enzimología , Conformación de Ácido Nucleico , Fosfatos/química , Fosfatos/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Multimerización de Proteína , Estructura Secundaria de Proteína , ARN/genética , ARN/metabolismo , ARN Catalítico/genética , ARN Catalítico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Spinach RNA aptamer contains a G-quadruplex motif that serves as a platform for binding and fluorescence activation of a GFP-like fluorophore. Here we show that Pb(2+) induces formation of Spinach's G-quadruplex and activates fluorescence with high selectivity and sensitivity. This device establishes the first example of an RNA-based sensor that provides a simple and inexpensive tool for Pb(2+) detection.