RESUMO
Biomolecules can be sequestered into membrane-less compartments, referred to as biomolecular condensates. Experimental and computational methods have helped define the physical-chemical properties of condensates. Less is known about how the high macromolecule concentrations in condensed phases contribute "solvent" interactions that can remodel the free-energy landscape of other condensate-resident proteins, altering thermally accessible conformations and, in turn, modulating function. Here, we use solution NMR spectroscopy to obtain atomic resolution insights into the interactions between the immature form of superoxide dismutase 1 (SOD1), which can mislocalize and aggregate in stress granules, and the RNA-binding protein CAPRIN1, a component of stress granules. NMR studies of CAPRIN1:SOD1 interactions, focused on both unfolded and folded SOD1 states in mixed phase and demixed CAPRIN1-based condensates, establish that CAPRIN1 shifts the SOD1 folding equilibrium toward the unfolded state through preferential interactions with the unfolded ensemble, with little change to the structure of the folded conformation. Key contacts between CAPRIN1 and the H80-H120 region of unfolded SOD1 are identified, as well as SOD1 interaction sites near both the arginine-rich and aromatic-rich regions of CAPRIN1. Unfolding of immature SOD1 in the CAPRIN1 condensed phase is shown to be coupled to aggregation, while a more stable zinc-bound, dimeric form of SOD1 is less susceptible to unfolding when solvated by CAPRIN1. Our work underscores the impact of the condensate solvent environment on the conformational states of resident proteins and supports the hypothesis that ALS mutations that decrease metal binding or dimerization function as drivers of aggregation in condensates.
Assuntos
Solventes , Superóxido Dismutase-1 , Superóxido Dismutase-1/química , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase-1/genética , Humanos , Solventes/química , Desdobramento de Proteína , Ligação Proteica , Dobramento de Proteína , Modelos Moleculares , Grânulos de Estresse/metabolismo , Grânulos de Estresse/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/química , Conformação Proteica , Espectroscopia de Ressonância MagnéticaRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/química , Conformação Molecular , Ácidos Nucleicos Heteroduplexes/química , Proteínas de Arabidopsis/química , Pareamento de Bases , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Termodinâmica , Fatores de Transcrição/químicaRESUMO
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.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Hibridização de Ácido Nucleico , Pareamento de Bases , DNA/genética , DNA/química , Conformação de Ácido Nucleico , RNA/químicaRESUMO
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.
Assuntos
Dano ao DNA , DNA , Humanos , Pareamento de Bases , DNA/química , MutagêneseRESUMO
Electrostatic interactions and charge balance are important for the formation of biomolecular condensates involving proteins and nucleic acids. However, a detailed, atomistic picture of the charge distribution around proteins during the phase-separation process is lacking. Here, we use solution NMR spectroscopy to measure residue-specific near-surface electrostatic potentials (ÏENS) of the positively charged carboxyl-terminal intrinsically disordered 103 residues of CAPRIN1, an RNA-binding protein localized to membraneless organelles playing an important role in messenger RNA (mRNA) storage and translation. Measured ÏENS values have been mapped along the adenosine triphosphate (ATP)-induced phase-separation trajectory. In the absence of ATP, ÏENS values for the mixed state of CAPRIN1 are positive and large and progressively decrease as ATP is added. This is coupled to increasing interchain interactions, particularly between aromatic-rich and arginine-rich regions of the protein. Upon phase separation, CAPRIN1 molecules in the condensed phase are neutral (ÏENS [Formula: see text] 0 mV), with â¼five molecules of ATP associated with each CAPRIN1 chain. Increasing the ATP concentration further inverts the CAPRIN1 electrostatic potential, so that molecules become negatively charged, especially in aromatic-rich regions, leading to re-entrance into a mixed phase. Our results collectively show that a subtle balance between electrostatic repulsion and interchain attractive interactions regulates CAPRIN1 phase separation and provides insight into how nucleotides, such as ATP, can induce formation of and subsequently dissolve protein condensates.
Assuntos
Fenômenos Bioquímicos , Proteínas Intrinsicamente Desordenadas , Transição de Fase , Proteínas de Ligação a RNA , Eletricidade Estática , Trifosfato de Adenosina/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Propriedades de SuperfícieRESUMO
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.
Assuntos
DNA , Conformação de Ácido Nucleico , RNA , Sequência de Bases , DNA/química , Congelamento , RNA/química , Termodinâmica , Raios UltravioletaRESUMO
Biomolecular condensates concentrate proteins, nucleic acids, and small molecules and play an essential role in many biological processes. Their formation is tuned by a balance between energetically favorable and unfavorable contacts, with charge-charge interactions playing a central role in some systems. The positively charged intrinsically disordered carboxy-terminal region of the RNA-binding protein CAPRIN1 is one such example, phase separating upon addition of negatively charged ATP or high concentrations of sodium chloride (NaCl). Using solution NMR spectroscopy, we measured residue-specific near-surface electrostatic potentials (ÏENS) of CAPRIN1 along its NaCl-induced phase separation trajectory to compare with those obtained using ATP. In both cases, electrostatic shielding decreases ÏENS values, yet surface potentials of CAPRIN1 in the two condensates can be different, depending on the amount of NaCl or ATP added. Our results establish that even small differences in ÏENS can significantly affect the level of protein enrichment and the mechanical properties of the condensed phase, leading, potentially, to the regulation of biological processes.
Assuntos
Hidrodinâmica , Proteínas Intrinsicamente Desordenadas , Proteínas de Ligação a RNA , Trifosfato de Adenosina , Proteínas Intrinsicamente Desordenadas/química , Proteínas de Ligação a RNA/química , Cloreto de Sódio/metabolismo , Eletricidade EstáticaRESUMO
NMR spectroscopy is an important tool for the measurement of the electrostatic properties of biomolecules. To this point, paramagnetic relaxation enhancements (PREs) of 1H nuclei arising from nitroxide cosolutes in biomolecular solutions have been used to measure effective near-surface electrostatic potentials (ÏENS) of proteins and nucleic acids. Here, we present a gadolinium (Gd)-based NMR method, exploiting Gd chelates with different net charges, for measuring ÏENS values and demonstrate its utility through applications to a number of biomolecular systems. The use of Gd-based cosolutes offers several advantages over nitroxides for ÏENS measurements. First, unlike nitroxide compounds, Gd chelates enable electrostatic potential measurements on oxidation-sensitive proteins that require reducing agents. Second, the large electron spin quantum number of Gd (7/2) results in notably larger PREs for Gd chelates when used at the same concentrations as nitroxide radicals. Thus, it is possible to measure ÏENS values exclusively from + and - charged compounds even for highly charged biomolecules, avoiding the use of neutral cosolutes that, as we further establish here, limits the accuracy of the measured electrostatic potentials. In addition, the smaller concentrations of cosolutes required minimize potential binding to sites on macromolecules. Fourth, the closer proximity of the paramagnetic center and charged groups within Gd chelates, in comparison to the corresponding nitroxide compounds, enables more accurate predictions of ÏENS potentials for cross-validation of the experimental results. The Gd-based method described here, thus, broadens the applicability of studies of biomolecular electrostatics using solution NMR spectroscopy.
Assuntos
Gadolínio , Eletricidade Estática , Gadolínio/química , Proteínas/química , Propriedades de Superfície , Ressonância Magnética Nuclear Biomolecular , Quelantes/química , Espectroscopia de Ressonância Magnética/métodosRESUMO
Biomolecular condensates can influence cellular function in a number of ways, including by changing the structural dynamics and conformational equilibria of the molecules partitioned within them. Here we use methyl transverse relaxation optimized spectroscopy (methyl-TROSY) NMR in conjunction with 2'-O-methyl labeling of RNA to characterize the thermodynamics and kinetics of RNA-RNA base pairing in condensates formed by the C-terminal intrinsically disordered region of CAPRIN1, an RNA-binding protein involved in RNA transport, translation, and stability. CAPRIN1 condensates destabilize RNA-RNA base pairing, resulting from a â¼270-fold decrease and a concomitant â¼15-fold increase in the on- and off-rates for duplex formation, respectively. The â¼30-fold slower diffusion of RNA single strands within the condensed phase partially accounts for the reduced on-rate, but the further â¼9-fold reduction likely reflects shedding of CAPRIN1 chains that are interacting with the RNA prior to hybridization. Our study emphasizes the important role of protein solvation in modulating nucleic acid recognition processes inside condensates.
Assuntos
Hibridização de Ácido Nucleico , RNA , Termodinâmica , RNA/química , Cinética , Conformação de Ácido Nucleico , Pareamento de Bases , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Separação de FasesRESUMO
It has recently been demonstrated that accurate near surface electrostatic potentials can be calculated for proteins from solvent paramagnetic relaxation enhancements (PREs) of amide protons measured using spin labels of similar structures but different charges (Yu et al. in Proc Natl Acad Sci 118(25):e2104020118, 2021). Here we develop methodology for extending such measurements to intrinsically disordered proteins at neutral pH where amide spectra are of very poor quality. Under these conditions it is shown that accurate PRE values can be measured using the haCONHA experiment that has been modified for recording 1Hα transverse relaxation rates. The optimal pulse scheme includes a spin-lock relaxation element for suppression of homonuclear scalar coupled evolution for all 1Hα protons, except those derived from Ser and Thr residues, and minimizes the radiation damping field from water magnetization that would otherwise increase measured relaxation rates. The robustness of the experiment is verified by developing a second approach using a band selective adiabatic decoupling scheme for suppression of scalar coupling modulations during 1Hα relaxation and showing that the measured PRE values from the two methods are in excellent agreement. The near surface electrostatic potential of a 103-residue construct comprising the C-terminal intrinsically disordered region of the RNA-binding protein CAPRIN1 is obtained at pH 5.5 using both 1HN and 1Hα-based relaxation rates, and at pH 7.4 where only 1Hα rates can be quantified, with very good agreement between potentials obtained under all experimental conditions.
Assuntos
Proteínas Intrinsicamente Desordenadas , Prótons , Amidas/química , Proteínas Intrinsicamente Desordenadas/química , Ressonância Magnética Nuclear Biomolecular/métodos , SolventesRESUMO
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.
Assuntos
Repetição Terminal Longa de HIV , Magnésio/química , Processamento Pós-Transcricional do RNA , RNA Viral/química , Pareamento de Bases , Cátions Bivalentes , Teoria da Densidade Funcional , HIV-1/química , Magnésio/metabolismo , Espectroscopia de Ressonância Magnética , Metilação , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Viral/genética , RNA Viral/metabolismo , TermodinâmicaRESUMO
DNA bases can adopt energetically unfavorable tautomeric forms that enable the formation of Watson-Crick-like (WC-like) mispairs, which have been proposed to give rise to spontaneous mutations in DNA and misincorporation errors in DNA replication and translation. Previous NMR and computational studies have indicated that the population of WC-like guanine-thymine (G-T) mispairs depends on the environment, such as the local nucleic acid sequence and solvation. To investigate these environmental effects, herein G-T mispair tautomerization processes are studied computationally in aqueous solution, in A-form and B-form DNA duplexes, and within the active site of a DNA polymerase λ variant. The wobble G-T (wG-T), WC-like G-T*, and WC-like G*-T forms are considered, where * indicates the enol tautomer of the base. The minimum free energy paths for the tautomerization from the wG-T to the WC-like G-T* and from the WC-like G-T* to the WC-like G*-T are computed with mixed quantum mechanical/molecular mechanical (QM/MM) free energy simulations. The reaction free energies and free energy barriers are found to be significantly influenced by the environment. The wG-TâG-T* tautomerization is predicted to be endoergic in aqueous solution and the DNA duplexes but slightly exoergic in the polymerase, with Arg517 and Asn513 providing electrostatic stabilization of G-T*. The G-T*âG*-T tautomerization is also predicted to be slightly more thermodynamically favorable in the polymerase relative to these DNA duplexes. These simulations are consistent with an experimentally driven kinetic misincorporation model suggesting that G-T mispair tautomerization occurs in the ajar polymerase conformation or concertedly with the transition from the ajar to the closed polymerase conformation. Furthermore, the order of the associated two proton transfer reactions is predicted to be different in the polymerase than in aqueous solution and the DNA duplexes. These studies highlight the impact of the environment on the thermodynamics, kinetics, and fundamental mechanisms of G-T mispair tautomerization, which plays a role in a wide range of biochemically important processes.
Assuntos
DNA Forma A/química , DNA de Forma B/química , Pareamento Incorreto de Bases , Pareamento de Bases , Domínio Catalítico , DNA Polimerase beta/química , DNA Forma A/genética , DNA de Forma B/genética , Guanina/química , Isomerismo , Modelos Moleculares , Teoria Quântica , Termodinâmica , Timina/químicaRESUMO
NMR off-resonance R1ρ relaxation dispersion measurements on base carbon and nitrogen nuclei have revealed that wobble G·T/U mismatches in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-abundance, and mutagenic Watson-Crick-like conformations. As Watson-Crick-like G·T mismatches have base pairing geometries similar to Watson-Crick base pairs, we hypothesized that they would mimic Watson-Crick base pairs with respect to the sugar-backbone conformation as well. Using off-resonance R1ρ measurements targeting the sugar C3' and C4' nuclei, a structure survey, and molecular dynamics simulations, we show that wobble G·T mismatches adopt sugar-backbone conformations that deviate from the canonical Watson-Crick conformation and that transitions toward tautomeric and anionic Watson-Crick-like G·T mismatches restore the canonical Watson-Crick sugar-backbone. These measurements also reveal kinetic isotope effects for tautomerization in D2O versus H2O, which provide experimental evidence in support of a transition state involving proton transfer. The results provide additional evidence in support of mutagenic Watson-Crick-like G·T mismatches, help rule out alternative inverted wobble conformations in the case of anionic G·T-, and also establish sugar carbons as new non-exchangeable probes of this exchange process.
Assuntos
Pareamento Incorreto de Bases , Carbono/química , DNA/química , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Açúcares/química , Pareamento de Bases , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Molecular , TiminaRESUMO
A(syn)-U/T and G(syn)-C+ Hoogsteen (HG) base pairs (bps) are energetically more disfavored relative to Watson-Crick (WC) bps in A-RNA as compared to B-DNA by >1 kcal/mol for reasons that are not fully understood. Here, we used NMR spectroscopy, optical melting experiments, molecular dynamics simulations and modified nucleotides to identify factors that contribute to this destabilization of HG bps in A-RNA. Removing the 2'-hydroxyl at single purine nucleotides in A-RNA duplexes did not stabilize HG bps relative to WC. In contrast, loosening the A-form geometry using a bulge in A-RNA reduced the energy cost of forming HG bps at the flanking sites to B-DNA levels. A structural and thermodynamic analysis of purine-purine HG mismatches reveals that compared to B-DNA, the A-form geometry disfavors syn purines by 1.5-4 kcal/mol due to sugar-backbone rearrangements needed to sterically accommodate the syn base. Based on MD simulations, an additional penalty of 3-4 kcal/mol applies for purine-pyrimidine HG bps due to the higher energetic cost associated with moving the bases to form hydrogen bonds in A-RNA versus B-DNA. These results provide insights into a fundamental difference between A-RNA and B-DNA duplexes with important implications for how they respond to damage and post-transcriptional modifications.
Assuntos
Pareamento de Bases/fisiologia , DNA de Forma B/química , Conformação de Ácido Nucleico , Purinas/química , RNA/química , DNA/química , Metabolismo Energético , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Pirimidinas/química , TermodinâmicaRESUMO
Biomolecules undergo motions on the micro-to-millisecond timescale to adopt low-populated transient states that play important roles in folding, recognition, and catalysis. NMR techniques, such as Carr-Purcell-Meiboom-Gill (CPMG), chemical exchange saturation transfer (CEST), and R1ρ are the most commonly used methods for characterizing such transitions at atomic resolution under solution conditions. CPMG and CEST are most effective at characterizing motions on the millisecond timescale. While some implementations of the R1ρ experiment are more broadly sensitive to motions on the micro-to-millisecond timescale, they entail the use of selective irradiation schemes and inefficient 1D data acquisition methods. Herein, we show that high-power radio-frequency fields can be used in CEST experiments to extend the sensitivity to faster motions on the micro-to-millisecond timescale. Given the ease of implementing high-power fields in CEST, this should make it easier to characterize micro-to-millisecond dynamics in biomolecules.
Assuntos
DNA/química , Espectroscopia de Ressonância Magnética/métodos , Ondas de Rádio , Limite de Detecção , Movimento (Física)RESUMO
N6-Methyladenosine (m6A) is an abundant epitranscriptomic modification that plays important roles in many aspects of RNA metabolism. While m6A is thought to mainly function by recruiting reader proteins to specific RNA sites, the modification can also reshape RNA-protein and RNA-RNA interactions by altering RNA structure mainly by destabilizing base pairing. Little is known about how m6A and other epitranscriptomic modifications might affect the kinetic rates of RNA folding and other conformational transitions that are also important for cellular activity. Here, we used NMR R1ρ relaxation dispersion and chemical exchange saturation transfer to noninvasively and site-specifically measure nucleic acid hybridization kinetics. The methodology was validated on two DNA duplexes and then applied to examine how a single m6A alters the hybridization kinetics in two RNA duplexes. The results show that m6A minimally impacts the rate constant for duplex dissociation, changing koff by â¼1-fold but significantly slows the rate of duplex annealing, decreasing kon by â¼7-fold. A reduction in the annealing rate was observed robustly for two different sequence contexts at different temperatures, both in the presence and absence of Mg2+. We propose that rotation of the N6-methyl group from the preferred syn conformation in the unpaired nucleotide to the energetically disfavored anti conformation required for Watson-Crick pairing is responsible for the reduced annealing rate. The results help explain why in mRNA m6A slows down tRNA selection and more generally suggest that m6A may exert cellular functions by reshaping the kinetics of RNA conformational transitions.
Assuntos
Adenosina/análogos & derivados , Ressonância Magnética Nuclear Biomolecular , RNA/química , Adenosina/análise , Adenosina/metabolismo , RNA/metabolismoRESUMO
In the canonical DNA double helix, Watson-Crick (WC) base pairs (bps) exist in dynamic equilibrium with sparsely populated (â¼0.02-0.4%) and short-lived (lifetimes â¼0.2-2.5 ms) Hoogsteen (HG) bps. To gain insights into transient HG bps, we used solution-state nuclear magnetic resonance spectroscopy, including measurements of residual dipolar couplings and molecular dynamics simulations, to examine how a single HG bp trapped using the N1-methylated adenine (m1A) lesion affects the structural and dynamic properties of two duplexes. The solution structure and dynamic ensembles of the duplexes reveals that in both cases, m1A forms a m1Aâ¢T HG bp, which is accompanied by local and global structural and dynamic perturbations in the double helix. These include a bias toward the BI backbone conformation; sugar repuckering, major-groove directed kinking (â¼9°); and local melting of neighboring WC bps. These results provide atomic insights into WC/HG breathing dynamics in unmodified DNA duplexes as well as identify structural and dynamic signatures that could play roles in m1A recognition and repair.
Assuntos
Adenina/química , Pareamento de Bases , Reparo do DNA , DNA/química , Conformação de Ácido Nucleico , Espectroscopia de Ressonância Magnética , Metilação , Soluções , Termodinâmica , Fatores de TempoRESUMO
NMR relaxation dispersion studies indicate that in canonical duplex DNA, Watson-Crick base pairs (bps) exist in dynamic equilibrium with short-lived low abundance excited state Hoogsteen bps. N1-methylated adenine (m1A) and guanine (m1G) are naturally occurring forms of damage that stabilize Hoogsteen bps in duplex DNA. NMR dynamic ensembles of DNA duplexes with m1A-T Hoogsteen bps reveal significant changes in sugar pucker and backbone angles in and around the Hoogsteen bp, as well as kinking of the duplex towards the major groove. Whether these structural changes also occur upon forming excited state Hoogsteen bps in unmodified duplexes remains to be established because prior relaxation dispersion probes provided limited information regarding the sugar-backbone conformation. Here, we demonstrate measurements of C3' and C4' spin relaxation in the rotating frame (R1ρ) in uniformly 13C/15N labeled DNA as sensitive probes of the sugar-backbone conformation in DNA excited states. The chemical shifts, combined with structure-based predictions using an automated fragmentation quantum mechanics/molecular mechanics method, show that the dynamic ensemble of DNA duplexes containing m1A-T Hoogsteen bps accurately model the excited state Hoogsteen conformation in two different sequence contexts. Formation of excited state A-T Hoogsteen bps is accompanied by changes in sugar-backbone conformation that allow the flipped syn adenine to form hydrogen-bonds with its partner thymine and this in turn results in overall kinking of the DNA toward the major groove. Results support the assignment of Hoogsteen bps as the excited state observed in canonical duplex DNA, provide an atomic view of DNA dynamics linked to formation of Hoogsteen bps, and lay the groundwork for a potentially general strategy for solving structures of nucleic acid excited states.
Assuntos
Adenina/química , Pareamento de Bases , Ressonância Magnética Nuclear Biomolecular/métodos , Timina/química , DNA/química , Ligação de Hidrogênio , Estrutura Molecular , Mutagênese , Conformação de Ácido NucleicoRESUMO
Watson-Crick like G-U mismatches with tautomeric Genol or Uenol bases can evade fidelity checkpoints and thereby contribute to translational errors. The 5-oxyacetic acid uridine (cmo5 U) modification is a base modification at the wobble position on tRNAs and is presumed to expand the decoding capability of tRNA at this position by forming Watson-Crick like cmo5 Uenol -G mismatches. A detailed investigation on the influence of the cmo5 U modification on structural and dynamic features of RNA was carried out by using solution NMR spectroscopy and UV melting curve analysis. The introduction of a stable isotope labeled variant of the cmo5 U modifier allowed the application of relaxation dispersion NMR to probe the potentially formed Watson-Crick like cmo5 Uenol -G base pair. Surprisingly, we find that at neutral pH, the modification promotes transient formation of anionic Watson-Crick like cmo5 U- -G, and not enolic base pairs. Our results suggest that recoding is mediated by an anionic Watson-Crick like species, as well as bring an interesting aspect of naturally occurring RNA modifications into focus-the fine tuning of nucleobase properties leading to modulation of the RNA structural landscape by adoption of alternative base pairing patterns.
RESUMO
Solution NMR spectroscopy has tremendous potential for providing atomic resolution insights into the interactions between proteins and nucleic acids partitioned into condensed phases of phase-separated systems. However, the highly viscous nature of the condensed phase challenges applications, and in particular, the extraction of quantitative, site-specific information. Here, we present a delayed decoupling-based HMQC pulse sequence for methyl-TROSY studies of 'client' proteins and nucleic acids partitioned into 'scaffold' proteinaceous phase-separated solvents. High sensitivity and excellent quality spectra are recorded of a nascent form of superoxide dismutase and of a small RNA fragment partitioned into CAPRIN1 condensates.