RESUMEN
RNAs fold into 3D structures that range from simple helical elements to complex tertiary structures and quaternary ribonucleoprotein assemblies. The functions of many regulatory RNAs depend on how their 3D structure changes in response to a diverse array of cellular conditions. In this Review, we examine how the structural characterization of RNA as dynamic ensembles of conformations, which form with different probabilities and at different timescales, is improving our understanding of RNA function in cells. We discuss the mechanisms of gene regulation by microRNAs, riboswitches, ribozymes, post-transcriptional RNA modifications and RNA-binding proteins, and how the cellular environment and processes such as liquid-liquid phase separation may affect RNA folding and activity. The emerging RNA-ensemble-function paradigm is changing our perspective and understanding of RNA regulation, from in vitro to in vivo and from descriptive to predictive.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Pliegue del ARN/fisiología , Procesamiento Postranscripcional del ARN/fisiología , ARN Catalítico , Proteínas de Unión al ARN , Animales , Humanos , ARN Catalítico/genética , ARN Catalítico/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
A key effector route of the Sugar Code involves lectins that exert crucial regulatory controls by targeting distinct cellular glycans. We demonstrate that a single amino-acid substitution in a banana lectin, replacing histidine 84 with a threonine, significantly reduces its mitogenicity, while preserving its broad-spectrum antiviral potency. X-ray crystallography, NMR spectroscopy, and glycocluster assays reveal that loss of mitogenicity is strongly correlated with loss of pi-pi stacking between aromatic amino acids H84 and Y83, which removes a wall separating two carbohydrate binding sites, thus diminishing multivalent interactions. On the other hand, monovalent interactions and antiviral activity are preserved by retaining other wild-type conformational features and possibly through unique contacts involving the T84 side chain. Through such fine-tuning, target selection and downstream effects of a lectin can be modulated so as to knock down one activity, while preserving another, thus providing tools for therapeutics and for understanding the Sugar Code.
Asunto(s)
Lectinas de Plantas/química , Lectinas de Plantas/genética , Fármacos Anti-VIH/química , Secuencia de Carbohidratos , Ingeniería Genética , Mitógenos/química , Modelos Moleculares , Simulación de Dinámica Molecular , Musa/químicaRESUMEN
RNA dynamics play a fundamental role in many cellular functions. However, there is no general framework to describe these complex processes, which typically consist of many structural maneuvers that occur over timescales ranging from picoseconds to seconds. Here, we classify RNA dynamics into distinct modes representing transitions between basins on a hierarchical free-energy landscape. These transitions include large-scale secondary-structural transitions at >0.1-s timescales, base-pair/tertiary dynamics at microsecond-to-millisecond timescales, stacking dynamics at timescales ranging from nanoseconds to microseconds, and other "jittering" motions at timescales ranging from picoseconds to nanoseconds. We review various modes within these three different tiers, the different mechanisms by which they are used to regulate function, and how they can be coupled together to achieve greater functional complexity.
Asunto(s)
Conformación de Ácido Nucleico , ARN/química , Emparejamiento Base , Técnicas Genéticas , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Movimiento (Física) , Conformación Proteica , Proteínas/química , Temperatura , TermodinámicaRESUMEN
Cellular processes are the product of interactions between biomolecules, which associate to form biologically active complexes1. These interactions are mediated by intermolecular contacts, which if disrupted, lead to alterations in cell physiology. Nevertheless, the formation of intermolecular contacts nearly universally requires changes in the conformations of the interacting biomolecules. As a result, binding affinity and cellular activity crucially depend both on the strength of the contacts and on the inherent propensities to form binding-competent conformational states2,3. Thus, conformational penalties are ubiquitous in biology and must be known in order to quantitatively model binding energetics for protein and nucleic acid interactions4,5. However, conceptual and technological limitations have hindered our ability to dissect and quantitatively measure how conformational propensities affect cellular activity. Here we systematically altered and determined the propensities for forming the protein-bound conformation of HIV-1 TAR RNA. These propensities quantitatively predicted the binding affinities of TAR to the RNA-binding region of the Tat protein and predicted the extent of HIV-1 Tat-dependent transactivation in cells. Our results establish the role of ensemble-based conformational propensities in cellular activity and reveal an example of a cellular process driven by an exceptionally rare and short-lived RNA conformational state.
Asunto(s)
Duplicado del Terminal Largo de VIH , VIH-1 , Conformación de Ácido Nucleico , ARN Viral , Activación Transcripcional , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Duplicado del Terminal Largo de VIH/genética , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , VIH-1/genética , VIH-1/metabolismoRESUMEN
Transcription factors recognize specific genomic sequences to regulate complex gene-expression programs. Although it is well-established that transcription factors bind to specific DNA sequences using a combination of base readout and shape recognition, some fundamental aspects of protein-DNA binding remain poorly understood1,2. Many DNA-binding proteins induce changes in the structure of the DNA outside the intrinsic B-DNA envelope. However, how the energetic cost that is associated with distorting the DNA contributes to recognition has proven difficult to study, because the distorted DNA exists in low abundance in the unbound ensemble3-9. Here we use a high-throughput assay that we term SaMBA (saturation mismatch-binding assay) to investigate the role of DNA conformational penalties in transcription factor-DNA recognition. In SaMBA, mismatched base pairs are introduced to pre-induce structural distortions in the DNA that are much larger than those induced by changes in the Watson-Crick sequence. Notably, approximately 10% of mismatches increased transcription factor binding, and for each of the 22 transcription factors that were examined, at least one mismatch was found that increased the binding affinity. Mismatches also converted non-specific sites into high-affinity sites, and high-affinity sites into 'super sites' that exhibit stronger affinity than any known canonical binding site. Determination of high-resolution X-ray structures, combined with nuclear magnetic resonance measurements and structural analyses, showed that many of the DNA mismatches that increase binding induce distortions that are similar to those induced by protein binding-thus prepaying some of the energetic cost incurred from deforming the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit transcription factors and thus modulate replication and repair activities in the cell10,11.
Asunto(s)
Proteínas de Unión al ADN/química , Conformación Molecular , Ácidos Nucleicos Heterodúplex/química , Proteínas de Arabidopsis/química , Emparejamiento Base , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutación , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Proteínas de Saccharomyces cerevisiae/química , Termodinámica , Factores de Transcripción/químicaRESUMEN
Many biochemical processes use the Watson-Crick geometry to distinguish correct from incorrect base pairing. However, on rare occasions, mismatches such as G·T/U can transiently adopt Watson-Crick-like conformations through tautomerization or ionization of the bases, giving rise to replicative and translational errors. The propensities to form Watson-Crick-like mismatches in RNA:DNA hybrids remain unknown, making it unclear whether they can also contribute to errors during processes such as transcription and CRISPR/Cas editing. Here, using NMR R1ρ experiments, we show that dG·rU and dT·rG mismatches in two RNA:DNA hybrids transiently form tautomeric (Genol·T/U $ \mathbin{\lower.3ex\hbox{$\buildrel\textstyle\rightarrow\over {\smash{\leftarrow}\vphantom{_{\vbox to.5ex{\vss}}}}$}}$ G·Tenol/Uenol) and anionic (G·T-/U-) Watson-Crick-like conformations. The tautomerization dynamics were like those measured in A-RNA and B-DNA duplexes. However, anionic dG·rU- formed with a ten-fold higher propensity relative to dT-·rG and dG·dT- and this could be attributed to the lower pKa (ΔpKa â¼0.4-0.9) of U versus T. Our findings suggest plausible roles for Watson-Crick-like G·T/U mismatches in transcriptional errors and CRISPR/Cas9 off-target gene editing, uncover a crucial difference between the chemical dynamics of G·U versus G·T, and indicate that anionic Watson-Crick-like G·U- could play a significant role evading Watson-Crick fidelity checkpoints in RNA:DNA hybrids and RNA duplexes.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Hibridación de Ácido Nucleico , Emparejamiento Base , ADN/genética , ADN/química , Conformación de Ácido Nucleico , ARN/químicaRESUMEN
In the mid-1930s, the English mathematician and logician Alan Turing invented an imaginary machine which could emulate the process of manipulating finite symbolic configurations by human computers. His machine launched the field of computer science and provided a foundation for the modern-day programmable computer. A decade later, building on Turing's machine, the American-Hungarian mathematician John von Neumann invented an imaginary self-reproducing machine capable of open-ended evolution. Through his machine, von Neumann answered one of the deepest questions in Biology: Why is it that all living organisms carry a self-description in the form of DNA? The story behind how two pioneers of computer science stumbled on the secret of life many years before the discovery of the DNA double helix is not well known, not even to biologists, and you will not find it in biology textbooks. Yet, the story is just as relevant today as it was eighty years ago: Turing and von Neumann left a blueprint for studying biological systems as if they were computing machines. This approach may hold the key to answering many remaining questions in Biology and could even lead to advances in computer science.
Asunto(s)
Marcha , Personal de Salud , HumanosRESUMEN
Replicative errors contribute to the genetic diversity needed for evolution but in high frequency can lead to genomic instability. Here, we show that DNA dynamics determine the frequency of misincorporating the Aâ¢G mismatch, and altered dynamics explain the high frequency of 8-oxoguanine (8OG) Aâ¢8OG misincorporation. NMR measurements revealed that Aantiâ¢Ganti (population (pop.) of >91%) transiently forms sparsely populated and short-lived Aanti+â¢Gsyn (pop. of ~2% and kex = kforward + kreverse of ~137 s-1) and Asynâ¢Ganti (pop. of ~6% and kex of ~2,200 s-1) Hoogsteen conformations. 8OG redistributed the ensemble, rendering Aantiâ¢8OGsyn the dominant state. A kinetic model in which Aanti+â¢Gsyn is misincorporated quantitatively predicted the dAâ¢dGTP misincorporation kinetics by human polymerase ß, the pH dependence of misincorporation and the impact of the 8OG lesion. Thus, 8OG increases replicative errors relative to G because oxidation of guanine redistributes the ensemble in favor of the mutagenic Aantiâ¢8OGsyn Hoogsteen state, which exists transiently and in low abundance in the Aâ¢G mismatch.
Asunto(s)
Daño del ADN , ADN , Humanos , Emparejamiento Base , ADN/química , MutagénesisRESUMEN
The majority of base pairs in double-stranded DNA exist in the canonical Watson-Crick geometry. However, they can also adopt alternate Hoogsteen conformations in various complexes of DNA with proteins and small molecules, which are key for biological function and mechanism. While detection of Hoogsteen base pairs in large DNA complexes and assemblies poses considerable challenges for traditional structural biology techniques, we show here that multidimensional dynamic nuclear polarization-enhanced solid-state NMR can serve as a unique spectroscopic tool for observing and distinguishing Watson-Crick and Hoogsteen base pairs in a broad range of DNA systems based on characteristic NMR chemical shifts and internuclear dipolar couplings. We illustrate this approach using a model 12-mer DNA duplex, free and in complex with the antibiotic echinomycin, which features two central adenine-thymine base pairs with Watson-Crick and Hoogsteen geometry, respectively, and subsequently extend it to the â¼200 kDa Widom 601 DNA nucleosome core particle.
Asunto(s)
Emparejamiento Base , ADN , Espectroscopía de Resonancia Magnética , Adenina/química , Adenina/metabolismo , ADN/química , Equinomicina/química , Espectroscopía de Resonancia Magnética/métodos , Timina/químicaRESUMEN
SignificanceUnderstanding and treating neurological disorders are global priorities. Some of these diseases are engendered by mutations that cause defects in the cellular synthesis of transfer RNAs (tRNAs), which function as adapter molecules that translate messenger RNAs into proteins. During tRNA biogenesis, ribonuclease P catalyzes removal of the transcribed sequence upstream of the mature tRNA. Here, we focus on a cytoplasmic tRNAArgUCU that is expressed specifically in neurons and, when harboring a particular point mutation, contributes to neurodegeneration in mice. Our results suggest that this mutation favors stable alternative structures that are not cleaved by mouse ribonuclease P and motivate a paradigm that may help to understand the molecular basis for disease-associated mutations in other tRNAs.
Asunto(s)
Homeostasis , Neuronas/metabolismo , Conformación de Ácido Nucleico , ARN de Transferencia/metabolismo , Animales , Emparejamiento Base , Corteza Cerebral/enzimología , Magnesio/metabolismo , Ratones , Modelos Moleculares , Mutación Puntual , Procesamiento Proteico-Postraduccional , ARN de Transferencia/química , ARN de Transferencia/genética , Ribonucleasa P/aislamiento & purificación , Ribonucleasa P/metabolismo , Especificidad por SustratoRESUMEN
Thermodynamic preferences to form non-native conformations are crucial for understanding how nucleic acids fold and function. However, they are difficult to measure experimentally because this requires accurately determining the population of minor low-abundance (<10%) conformations in a sea of other conformations. Here, we show that melting experiments enable facile measurements of thermodynamic preferences to adopt nonnative conformations in DNA and RNA. The key to this "delta-melt" approach is to use chemical modifications to render specific minor non-native conformations the major state. The validity and robustness of delta-melt is established for four different non-native conformations under various physiological conditions and sequence contexts through independent measurements of thermodynamic preferences using NMR. Delta-melt is faster relative to NMR, simple, and cost-effective and enables thermodynamic preferences to be measured for exceptionally low-populated conformations. Using delta-melt, we obtained rare insights into conformational cooperativity, obtaining evidence for significant cooperativity (1.0 to 2.5 kcal/mol) when simultaneously forming two adjacent Hoogsteen base pairs. We also measured the thermodynamic preferences to form G-C+ and A-T Hoogsteen and A-T base open states for nearly all 16 trinucleotide sequence contexts and found distinct sequence-specific variations on the order of 2 to 3 kcal/mol. This rich landscape of sequence-specific non-native minor conformations in the DNA double helix may help shape the sequence specificity of DNA biochemistry. Thus, melting experiments can now be used to access thermodynamic information regarding regions of the free energy landscape of biomolecules beyond the native folded and unfolded conformations.
Asunto(s)
ADN , Conformación de Ácido Nucleico , ARN , Secuencia de Bases , ADN/química , Congelación , ARN/química , Termodinámica , Rayos UltravioletaRESUMEN
Tautomeric and anionic Watson-Crick-like mismatches have important roles in replication and translation errors through mechanisms that are not fully understood. Here, using NMR relaxation dispersion, we resolve a sequence-dependent kinetic network connecting Gâ¢T/U wobbles with three distinct Watson-Crick mismatches: two rapidly exchanging tautomeric species (Genolâ¢T/UGâ¢Tenol/Uenol; population less than 0.4%) and one anionic species (Gâ¢T-/U-; population around 0.001% at neutral pH). The sequence-dependent tautomerization or ionization step was inserted into a minimal kinetic mechanism for correct incorporation during replication after the initial binding of the nucleotide, leading to accurate predictions of the probability of dGâ¢dT misincorporation across different polymerases and pH conditions and for a chemically modified nucleotide, and providing mechanisms for sequence-dependent misincorporation. Our results indicate that the energetic penalty for tautomerization and/or ionization accounts for an approximately 10-2 to 10-3-fold discrimination against misincorporation, which proceeds primarily via tautomeric dGenolâ¢dT and dGâ¢dTenol, with contributions from anionic dGâ¢dT- dominant at pH 8.4 and above or for some mutagenic nucleotides.
Asunto(s)
Disparidad de Par Base , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/biosíntesis , ADN/química , Guanina/metabolismo , Mutagénesis , Timina/metabolismo , Animales , Aniones , Disparidad de Par Base/genética , ADN/genética , Guanina/química , Humanos , Concentración de Iones de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Probabilidad , Ratas , Timina/químicaRESUMEN
Knowing the 3D structures formed by the various conformations populating the RNA free-energy landscape, their relative abundance, and kinetic interconversion rates is required to obtain a quantitative and predictive understanding of how RNAs fold and function at the atomic level. While methods integrating ensemble-averaged experimental data with computational modeling are helping define the most abundant conformations in RNA ensembles, elucidating their kinetic rates of interconversion and determining the 3D structures of sparsely populated short-lived RNA excited conformational states (ESs) remains challenging. Here, we developed an approach integrating Rosetta-FARFAR RNA structure prediction with NMR residual dipolar couplings and relaxation dispersion that simultaneously determines the 3D structures formed by the ground-state (GS) and ES subensembles, their relative abundance, and kinetic rates of interconversion. The approach is demonstrated on HIV-1 TAR, whose six-nucleotide apical loop was previously shown to form a sparsely populated (â¼13%) short-lived (lifetime â¼ 45 µs) ES. In the GS, the apical loop forms a broad distribution of open conformations interconverting on the pico-to-nanosecond time scale. Most residues are unpaired and preorganized to bind the Tat-superelongation protein complex. The apical loop zips up in the ES, forming a narrow distribution of closed conformations, which sequester critical residues required for protein recognition. Our work introduces an approach for determining the 3D ensemble models formed by sparsely populated RNA conformational states, provides a rare atomic view of an RNA ES, and kinetically resolves the atomic 3D structures of RNA conformational substates, interchanging on time scales spanning 6 orders of magnitude, from picoseconds to microseconds.
Asunto(s)
Proteínas , ARN , ARN/química , Resonancia Magnética Nuclear Biomolecular , Espectroscopía de Resonancia Magnética , Conformación de Ácido Nucleico , Proteínas/genéticaRESUMEN
Identifying small molecules that selectively bind an RNA target while discriminating against all other cellular RNAs is an important challenge in RNA-targeted drug discovery. Much effort has been directed toward identifying drug-like small molecules that minimize electrostatic and stacking interactions that lead to nonspecific binding of aminoglycosides and intercalators to many stem-loop RNAs. Many such compounds have been reported to bind RNAs and inhibit their cellular activities. However, target engagement and cellular selectivity assays are not routinely performed, and it is often unclear whether functional activity directly results from specific binding to the target RNA. Here, we examined the propensities of three drug-like compounds, previously shown to bind and inhibit the cellular activities of distinct stem-loop RNAs, to bind and inhibit the cellular activities of two unrelated HIV-1 stem-loop RNAs: the transactivation response element (TAR) and the rev response element stem IIB (RREIIB). All compounds bound TAR and RREIIB in vitro, and two inhibited TAR-dependent transactivation and RRE-dependent viral export in cell-based assays while also exhibiting off-target interactions consistent with nonspecific activity. A survey of X-ray and NMR structures of RNA-small molecule complexes revealed that aminoglycosides and drug-like molecules form hydrogen bonds with functional groups commonly accessible in canonical stem-loop RNA motifs, in contrast to ligands that specifically bind riboswitches. Our results demonstrate that drug-like molecules can nonspecifically bind stem-loop RNAs most likely through hydrogen bonding and electrostatic interactions and reinforce the importance of assaying for off-target interactions and RNA selectivity in vitro and in cells when assessing novel RNA-binders.
Asunto(s)
Aminoglicósidos/farmacología , Genes env/efectos de los fármacos , Duplicado del Terminal Largo de VIH/efectos de los fármacos , ARN Viral/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Aminoglicósidos/química , Aminoglicósidos/metabolismo , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Bioensayo , Descubrimiento de Drogas , VIH-1/efectos de los fármacos , VIH-1/genética , VIH-1/metabolismo , Humanos , Enlace de Hidrógeno , Isoquinolinas/química , Isoquinolinas/metabolismo , Isoquinolinas/farmacología , Conformación de Ácido Nucleico , Pentamidina/química , Pentamidina/metabolismo , Pentamidina/farmacología , ARN Viral/genética , ARN Viral/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Electricidad Estática , Activación Transcripcional/efectos de los fármacos , Yohimbina/química , Yohimbina/metabolismo , Yohimbina/farmacologíaRESUMEN
Watson-Crick base pairs (bps) are the fundamental unit of genetic information and the building blocks of the DNA double helix. However, A-T and G-C can also form alternative 'Hoogsteen' bps, expanding the functional complexity of DNA. We developed 'Hoog-finder', which uses structural fingerprints to rapidly screen Hoogsteen bps, which may have been mismodeled as Watson-Crick in crystal structures of protein-DNA complexes. We uncovered 17 Hoogsteen bps, 7 of which were in complex with 6 proteins never before shown to bind Hoogsteen bps. The Hoogsteen bps occur near mismatches, nicks and lesions and some appear to participate in recognition and damage repair. Our results suggest a potentially broad role for Hoogsteen bps in stressed regions of the genome and call for a community-wide effort to identify these bps in current and future crystal structures of DNA and its complexes.
Asunto(s)
Emparejamiento Base , Proteínas de Unión al ADN/química , ADN/química , Conformación de Ácido Nucleico , Dominios Proteicos , Secuencia de Bases , Sitios de Unión/genética , Biología Computacional/métodos , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Bases de Datos Genéticas , Enlace de Hidrógeno , Modelos Moleculares , Mutación , Unión Proteica , TermodinámicaRESUMEN
2'-O-Methyl (Nm) is a highly abundant post-transcriptional RNA modification that plays important biological roles through mechanisms that are not entirely understood. There is evidence that Nm can alter the biological activities of RNAs by biasing the ribose sugar pucker equilibrium toward the C3'-endo conformation formed in canonical duplexes. However, little is known about how Nm might more broadly alter the dynamic ensembles of flexible RNAs containing bulges and internal loops. Here, using NMR and the HIV-1 transactivation response (TAR) element as a model system, we show that Nm preferentially stabilizes alternative secondary structures in which the Nm-modified nucleotides are paired, increasing both the abundance and lifetime of low-populated short-lived excited states by up to 10-fold. The extent of stabilization increased with number of Nm modifications and was also dependent on Mg2+. Through phi-value analysis, the Nm modification also provided rare insights into the structure of the transition state for conformational exchange. Our results suggest that Nm could alter the biological activities of Nm-modified RNAs by modulating their secondary structural ensembles as well as establish the utility of Nm as a tool for the discovery and characterization of RNA excited state conformations.
Asunto(s)
Duplicado del Terminal Largo de VIH , Magnesio/química , Procesamiento Postranscripcional del ARN , ARN Viral/química , Emparejamiento Base , Cationes Bivalentes , Teoría Funcional de la Densidad , VIH-1/química , Magnesio/metabolismo , Espectroscopía de Resonancia Magnética , Metilación , Conformación de Ácido Nucleico , Estabilidad del ARN , ARN Viral/genética , ARN Viral/metabolismo , TermodinámicaRESUMEN
As the Watson-Crick faces of nucleobases are protected in dsDNA, it is commonly assumed that deleterious alkylation damage to the Watson-Crick faces of nucleobases predominantly occurs when DNA becomes single-stranded during replication and transcription. However, damage to the Watson-Crick faces of nucleobases has been reported in dsDNA in vitro through mechanisms that are not understood. In addition, the extent of protection from methylation damage conferred by dsDNA relative to ssDNA has not been quantified. Watson-Crick base pairs in dsDNA exist in dynamic equilibrium with Hoogsteen base pairs that expose the Watson-Crick faces of purine nucleobases to solvent. Whether this can influence the damage susceptibility of dsDNA remains unknown. Using dot-blot and primer extension assays, we measured the susceptibility of adenine-N1 to methylation by dimethyl sulfate (DMS) when in an A-T Watson-Crick versus Hoogsteen conformation. Relative to unpaired adenines in a bulge, Watson-Crick A-T base pairs in dsDNA only conferred â¼130-fold protection against adenine-N1 methylation, and this protection was reduced to â¼40-fold for A(syn)-T Hoogsteen base pairs embedded in a DNA-drug complex. Our results indicate that Watson-Crick faces of nucleobases are accessible to alkylating agents in canonical dsDNA and that Hoogsteen base pairs increase this accessibility. Given the higher abundance of dsDNA relative to ssDNA, these results suggest that dsDNA could be a substantial source of cytotoxic damage. The work establishes DMS probing as a method for characterizing A(syn)-T Hoogsteen base pairs in vitro and also lays the foundation for a sequencing approach to map A(syn)-T Hoogsteen and unpaired adenines genome-wide in vivo.
Asunto(s)
Emparejamiento Base , Metilación de ADN , ADN/química , Ésteres del Ácido Sulfúrico/químicaRESUMEN
Rare tautomeric and anionic nucleobases are believed to have fundamental biological roles, but their prevalence and functional importance has remained elusive because they exist transiently, in low abundance, and involve subtle movements of protons that are difficult to visualize. Using NMR relaxation dispersion, we show here that wobble dGâ¢dT and rGâ¢rU mispairs in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-populated Watson-Crick-like mispairs that are stabilized by rare enolic or anionic bases. These mispairs can evade Watson-Crick fidelity checkpoints and form with probabilities (10(-3) to 10(-5)) that strongly imply a universal role in replication and translation errors. Our results indicate that rare tautomeric and anionic bases are widespread in nucleic acids, expanding their structural and functional complexity beyond that attainable with canonical bases.
Asunto(s)
Emparejamiento Base , ADN/química , Ácidos Nucleicos Heterodúplex/química , ARN/química , Secuencia de Bases , Dermatoglifia del ADN , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Mutación/genética , ProbabilidadRESUMEN
The HIV-1 Rev response element (RRE) RNA element mediates the nuclear export of intron containing viral RNAs by forming an oligomeric complex with the viral protein Rev. Stem IIB and nearby stem II three-way junction nucleate oligomerization through cooperative binding of two Rev molecules. Conformational flexibility at this RRE region has been shown to be important for Rev binding. However, the nature of the flexibility has remained elusive. Here, using NMR relaxation dispersion, including a new strategy for directly observing transient conformational states in large RNAs, we find that stem IIB alone or when part of the larger RREII three-way junction robustly exists in dynamic equilibrium with non-native excited state (ES) conformations that have a combined population of â¼20%. The ESs disrupt the Rev-binding site by changing local secondary structure, and their stabilization via point substitution mutations decreases the binding affinity to the Rev arginine-rich motif (ARM) by 15- to 80-fold. The ensemble clarifies the conformational flexibility observed in stem IIB, reveals long-range conformational coupling between stem IIB and the three-way junction that may play roles in cooperative Rev binding, and also identifies non-native RRE conformational states as new targets for the development of anti-HIV therapeutics.