RESUMEN
The folding of linear polymers into discrete three-dimensional structures is often required for biological function. The formation of long-lived intermediates is a hallmark of the folding of large RNA molecules due to the ruggedness of their energy landscapes. The precise thermodynamic nature of the barriers (whether enthalpic or entropic) that leads to intermediate formation is still poorly characterized in large structured RNA molecules. A classic approach to analyzing kinetic barriers are temperature dependent studies analyzed with Eyring's transition state theory. We applied Eyring's theory to time-resolved hydroxyl radical (â¢OH) footprinting kinetics progress curves collected at eight temperature from 21.5 °C to 51 °C to characterize the thermodynamic nature of folding intermediate formation for the Mg(2+)-mediated folding of the Tetrahymena thermophila group I ribozyme. A common kinetic model configuration describes this RNA folding reaction over the entire temperature range studied consisting of primary (fast) transitions to misfolded intermediates followed by much slower secondary transitions, consistent with previous studies. Eyring analysis reveals that the primary transitions are moderate in magnitude and primarily enthalpic in nature. In contrast, the secondary transitions are daunting in magnitude and entropic in nature. The entropic character of the secondary transitions is consistent with structural rearrangement of the intermediate species to the final folded form. This segregation of kinetic control reveals distinctly different molecular mechanisms during the two stages of RNA folding and documents the importance of entropic barriers to defining rugged RNA folding landscapes.
Asunto(s)
Pliegue del ARN/genética , Secuencia de Bases , Radical Hidroxilo/metabolismo , Cinética , Magnesio/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN Protozoario/genética , Temperatura , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo , TermodinámicaRESUMEN
The complex formed between the U2 and U6 small nuclear (sn)RNA molecules of the eukaryotic spliceosome plays a critical role in the catalysis of precursor mRNA splicing. Here, we have used enzymatic structure probing, (19)F NMR, and analytical ultracentrifugation techniques to characterize the fold of a protein-free biophysically tractable paired construct representing the human U2-U6 snRNA complex. Results from enzymatic probing and (19)F NMR for the complex in the absence of Mg(2+) are consistent with formation of a four-helix junction structure as a predominant conformation. However, (19)F NMR data also identify a lesser fraction (up to 14% at 25°C) of a three-helix conformation. Based upon this distribution, the calculated ΔG for inter-conversion to the four-helix structure from the three-helix structure is approximately -4.6 kJ/mol. In the presence of 5 mM Mg(2+), the fraction of the three-helix conformation increased to â¼17% and the Stokes radius, measured by analytical ultracentrifugation, decreased by 2%, suggesting a slight shift to an alternative conformation. NMR measurements demonstrated that addition of an intron fragment to the U2-U6 snRNA complex results in displacement of U6 snRNA from the region of Helix III immediately 5' of the ACAGAGA sequence of U6 snRNA, which may facilitate binding of the segment of the intron adjacent to the 5' splice site to the ACAGAGA sequence. Taken together, these observations indicate conformational heterogeneity in the protein-free human U2-U6 snRNA complex consistent with a model in which the RNA has sufficient conformational flexibility to facilitate inter-conversion between steps of splicing in situ.
Asunto(s)
ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/metabolismo , Saccharomyces cerevisiae/genética , Empalmosomas/química , Secuencia de Bases , Metabolismo Energético , Humanos , Datos de Secuencia Molecular , Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismoRESUMEN
In RNA, function follows form. Mapping the surface of RNA molecules with chemical and enzymatic probes has revealed invaluable information about structure and folding. Hydroxyl radicals ((·)OH) map the surface of nucleic acids by cutting the backbone where it is accessible to solvent. Recent studies showed that a microfluidic chip containing pyrite (FeS(2)) can produce sufficient (·)OH to footprint DNA. The 49-nt Diels-Alder RNA enzyme catalyzes the C-C bond formation between a diene and a dienophile. A crystal structure, molecular dynamics simulation and atomic mutagenesis studies suggest that nucleotides of an asymmetric bulge participate in the dynamic architecture of the ribozyme's active center. Of note is that residue U42 directly interacts with the product in the crystallized RNA/product complex. Here, we use powdered pyrite held in a commercially available cartridge to footprint the Diels-Alderase ribozyme with single nucleotide resolution. Residues C39 to U42 are more reactive to (·)OH than predicted by the solvent accessibility calculated from the crystal structure suggesting that this loop is dynamic in solution. The loop's flexibility may contribute to substrate recruitment and product release. Our implementation of pyrite-mediated (·)OH footprinting is a readily accessible approach to gleaning information about the architecture of small RNA molecules.
Asunto(s)
Hierro/química , Pliegue del ARN , ARN Catalítico/química , Análisis de Secuencia de ARN/métodos , Sulfuros/químicaRESUMEN
RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling a specific biologic role as regulator, binder or catalyst. Information about tertiary contact formation is essential to understand the function of RNA molecules. Hydroxyl radicals (â¢OH) are unique probes of the structure of nucleic acids due to their high reactivity and small size. When used as a footprinting probe, hydroxyl radicals map the solvent accessible surface of the phosphodiester backbone of DNA and RNA with as fine as single nucleotide resolution. Hydroxyl radical footprinting can be used to identify the nucleotides within an intermolecular contact surface, e.g. in DNA-protein and RNA-protein complexes. Equilibrium and kinetic transitions can be determined by conducting hydroxyl radical footprinting as a function of a solution variable or time, respectively. A key feature of footprinting is that limited exposure to the probe (e.g., 'single-hit kinetics') results in the uniform sampling of each nucleotide of the polymer. In this video article, we use the P4-P6 domain of the Tetrahymena ribozyme to illustrate RNA sample preparation and the determination of a Mg(II)-mediated folding isotherms. We describe the use of the well known hydroxyl radical footprinting protocol that requires H(2)O(2) (we call this the 'peroxidative' protocol) and a valuable, but not widely known, alternative that uses naturally dissolved O(2)(we call this the 'oxidative' protocol). An overview of the data reduction, transformation and analysis procedures is presented.
Asunto(s)
Radical Hidroxilo/química , Peróxidos/química , ARN/química , Cationes Bivalentes/química , Peróxido de Hidrógeno/química , Cinética , Magnesio/química , Conformación de Ácido Nucleico , Oxidación-Reducción , ARN Catalítico/química , Tetrahymena/enzimologíaRESUMEN
We describe a microfluidic device containing a mineral matrix capable of rapidly generating hydroxyl radicals that enables high-resolution structural studies of nucleic acids. Hydroxyl radicals cleave the solvent accessible backbone of DNA and RNA; the cleavage products can be detected with as fine as single nucleotide resolution. Protection from hydroxyl radical cleavage (footprinting) can identify sites of protein binding or the presence of tertiary structure. Here we report preparation of micron sized particles of iron sulfide (pyrite) and fabrication of a microfluidic prototype that together generate enough hydroxyl radicals within 20 ms to cleave DNA sufficiently for a footprinting analysis to be conducted. This prototype enables the development of high-throughput and/or rapid reaction devices with which to probe nucleic acid folding dynamics and ligand binding.
Asunto(s)
ADN/química , Radical Hidroxilo/química , Técnicas Analíticas Microfluídicas/instrumentación , Solventes/química , Secuencia de Bases , ADN/genética , Datos de Secuencia Molecular , Espectrometría de Fluorescencia , Propiedades de Superficie , Factores de TiempoRESUMEN
A variety of analytical techniques are used to probe the mechanisms by which RNA molecules fold to discrete three dimensional structures. Methods such as small angle X-ray scattering (SAXS) report global properties like overall size and shape of the RNA. Other methods such as chemical or enzymatic mapping (footprinting) report properties with resolution as fine as single nucleotide. The hydroxyl radical (*OH) is a footprinting probe which cleaves the oligonucleotide backbone independently of sequence and thus is a valuable reporter of backbone solvent accessibility. Combinations of global and local measures of folding reactions are uniquely able to distinguish specific from nonspecific processes. This article highlights the application of *OH footprinting as a complement to SAXS for kinetics analysis of RNA folding. We illustrate this combination of techniques using a study of the role played by the stiffness of a hinge in determining the rate limiting step of a Mg(2+)-mediated RNA folding reaction.
Asunto(s)
ARN/química , Biofisica/métodos , Diseño de Equipo , Radical Hidroxilo , Cinética , Modelos Estadísticos , Conformación de Ácido Nucleico , Polímeros/química , ARN Catalítico/química , Dispersión de Radiación , Dispersión del Ángulo Pequeño , Solventes/química , Tetrahymena thermophila/metabolismo , Factores de Tiempo , Rayos XRESUMEN
Metal ions play a crucial role in the conformation and splicing activity of Group II introns. Results from 2-aminopurine fluorescence and solution NMR studies suggest that metal ion binding within the branch site region of native D6 of the Group II intron is specific for alkaline earth metal ions and involves inner sphere coordination. Although Mg(2+) and Ca(2+) still bind to a mutant stem loop sequence from which the internal loop had been deleted, ion binding to the mutant RNA results in decreased, rather than increased, exposure of the branch site residue to solvent. These data further support the role of the internal loop in defining branch site conformation of the Group II intron. The specific bound Mg(2+) may play a bivalent role: facilitates the extrahelical conformation of the branch site and has the potential to act as a Lewis acid during splicing.
Asunto(s)
Intrones/genética , Magnesio/metabolismo , Conformación de Ácido Nucleico , Espectroscopía de Resonancia Magnética , ARN/química , ARN/genética , Empalme del ARN , Especificidad por SustratoRESUMEN
Cation-mediated RNA folding from extended to compact, biologically active conformations relies on a temporal balance of forces. The Mg2 +-mediated folding of the Tetrahymena thermophila ribozyme is characterized by rapid nonspecific collapse followed by tertiary-contact-induced compaction. This article focuses on an autonomously folding portion of the Tetrahymena ribozyme, its P4-P6 domain, in order to probe one facet of the rapid collapse: chain flexibility. The time evolution of P4-P6 folding was followed by global and local measures as a function of Mg2 + concentration. While all concentrations of Mg2 + studied are sufficient to screen the charge on the helices, the rates of compaction and tertiary contact formation diverge as the concentration of Mg2 + increases; collapse is greatly accelerated by Mg2 +, while tertiary contact formation is not. These studies highlight the importance of chain stiffness to RNA folding; at 10 mM Mg2 +, a stiff hinge limits the rate of P4-P6 folding. At higher magnesium concentrations, the rate-limiting step shifts from hinge bending to tertiary contact formati
Asunto(s)
Conformación de Ácido Nucleico , ARN/química , Animales , Secuencia de Bases , Radical Hidroxilo/química , Cinética , Magnesio/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico/efectos de los fármacos , Concentración Osmolar , ARN Catalítico/química , ARN Catalítico/genética , ARN Protozoario/química , ARN Protozoario/genética , Dispersión del Ángulo Pequeño , Tetrahymena thermophila/química , Tetrahymena thermophila/enzimología , Tetrahymena thermophila/genética , Difracción de Rayos XRESUMEN
Simple lysine conjugates are capable of selective DNA damage at sites approximating a variety of naturally occurring DNA-damage patterns. This process transforms single-strand DNA cleavage into double-strand cleavage with a potential impact on gene and cancer therapy or on the design of DNA constructs that require disassembly at a specific location. This study constitutes an example of DNA damage site recognition by molecules that are two orders of magnitude smaller than DNA-processing enzymes and presents a strategy for site-selective cleavage of single-strand nucleotides, which is based on their annealing with two shorter counterstrands designed to recreate the above duplex damage site.
Asunto(s)
Daño del ADN , Lisina/química , ADN de Cadena Simple/químicaRESUMEN
We report the chemical synthesis of 5'-amino- and 5'-thiol-hexaethylene glycol guanosine nucleotides and their enzymatic incorporation into RNA, followed by chemical modifications at their nucleophilic ends. By using two similar routes, the conjugates of guanosine-5'-monophosphate and hexaethylene glycol with attached reactive groups (SH or NH(2)) were synthesized using phosphoramidite chemistry, and characterized by MALDI TOF mass spectrometry. These initiator molecules were efficiently incorporated into RNA at the 5'-end by run-off transcription using T7 RNA polymerase. The potential of these RNA conjugates for a broad reaction range with electrophiles is shown here, thereby enabling their use for diverse biochemical applications.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/química , Nucleótidos/química , ARN/química , Compuestos de Sulfhidrilo/química , Proteínas Virales/química , Codón Iniciador , ARN Catalítico/químicaRESUMEN
Group II introns are multidomain ribozymes that catalyze their own removal from pre-mRNA. The nucleophile for the first cleavage step is the 2'OH of a specific adenosine within domain 6 (D6), called the branch site. Mechanistic parallels and limited secondary structural similarity with the eukaryotic spliceosome lead many to speculate that the two systems have a common ancestry. We have elucidated structural features of the branch site region and the importance of the internal loop to branch site conformation within D6 of the ai5gamma Group II intron by NMR and fluorescence spectroscopy. Fluorescence experiments in which 2-aminopurine was substituted for the branch site adenosine suggest that the branch site base is exposed to solvent and that this position is enhanced by Mg2+ or Ca2+. Upfield NMR chemical shifts of imino protons of the two uridine residues flanking the branch site adenosine, and an n --> n + 2 NOE between them, suggest a stacked intrahelical conformation of the two uridines. In contrast, results of NMR and 2-aminopurine fluorescence spectra of a mutated D6 from which the internal loop had been deleted suggest a less exposed position of the branch site adenosine, which is likely to form a G-A base pair with the opposing 3'G. These findings describe a model in which the branch site adenosine of D6 is in an extrahelical position, surrounded by two intrahelical bases. The internal loop and divalent metal ions facilitate this motif.
Asunto(s)
Intrones/genética , Autoempalme del ARN Ribosómico/química , 2-Aminopurina/química , Adenosina/química , Secuencia de Bases , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación de Ácido Nucleico , Autoempalme del ARN Ribosómico/genética , SolucionesRESUMEN
Development of artificial ribozymes by in vitro selection has so far, mostly been addressed from the viewpoint of fundamental research. However, such ribozymes also have high potential as selective catalysts in practical syntheses. Immobilization of an active and selective ribozyme is an important step towards this end. A 49-nucleotide RNA molecule that was previously found to stereoselectively catalyze Diels-Alder reactions between various anthracene dienes and maleimide dienophiles was quantitatively immobilized on an agarose matrix by periodate oxidation of the 3'-terminal ribose and coupling to a hydrazide moiety. Typical loadings were 45 pmol microL(-1) gel. The specific activity was comparable to that of soluble ribozyme, and high enantioselectivities were obtained in catalyzed cycloadditions. The catalytic matrix was found to be stable and could be regenerated about 40 times with only minimal reduction of catalytic activity. Like the soluble ribozyme, the immobilized catalyst stereoselectively converts various diene and dienophile substrates. By using either natural D-RNA or enantiomeric L-RNA, both product enantiomers were made synthetically accessible with similar selectivities.