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1.
Sci Adv ; 9(39): eadj3509, 2023 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-37756398

RESUMO

RNA macromolecules, like proteins, fold to assume shapes that are intimately connected to their broadly recognized biological functions; however, because of their high charge and dynamic nature, RNA structures are far more challenging to determine. We introduce an approach that exploits the high brilliance of x-ray free-electron laser sources to reveal the formation and ready identification of angstrom-scale features in structured and unstructured RNAs. Previously unrecognized structural signatures of RNA secondary and tertiary structures are identified through wide-angle solution scattering experiments. With millisecond time resolution, we observe an RNA fold from a dynamically varying single strand through a base-paired intermediate to assume a triple-helix conformation. While the backbone orchestrates the folding, the final structure is locked in by base stacking. This method may help to rapidly characterize and identify structural elements in nucleic acids in both equilibrium and time-resolved experiments.


Assuntos
Ácidos Nucleicos , RNA , Elétrons , Lasers
2.
bioRxiv ; 2023 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-37292849

RESUMO

RNA macromolecules, like proteins, fold to assume shapes that are intimately connected to their broadly recognized biological functions; however, because of their high charge and dynamic nature, RNA structures are far more challenging to determine. We introduce an approach that exploits the high brilliance of x-ray free electron laser sources to reveal the formation and ready identification of Å scale features in structured and unstructured RNAs. New structural signatures of RNA secondary and tertiary structures are identified through wide angle solution scattering experiments. With millisecond time resolution, we observe an RNA fold from a dynamically varying single strand through a base paired intermediate to assume a triple helix conformation. While the backbone orchestrates the folding, the final structure is locked in by base stacking. In addition to understanding how RNA triplexes form and thereby function as dynamic signaling elements, this new method can vastly increase the rate of structure determination for these biologically essential, but mostly uncharacterized macromolecules.

3.
IUCrJ ; 10(Pt 3): 363-375, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-37144817

RESUMO

Advances in time-resolved structural techniques, mainly in macromolecular crystallography and small-angle X-ray scattering (SAXS), allow for a detailed view of the dynamics of biological macromolecules and reactions between binding partners. Of particular promise, are mix-and-inject techniques, which offer a wide range of experimental possibility as microfluidic mixers are used to rapidly combine two species just prior to data collection. Most mix-and-inject approaches rely on diffusive mixers, which have been effectively used within crystallography and SAXS for a variety of systems, but their success is dependent on a specific set of conditions to facilitate fast diffusion for mixing. The use of a new chaotic advection mixer designed for microfluidic applications helps to further broaden the types of systems compatible with time-resolved mixing experiments. The chaotic advection mixer can create ultra-thin, alternating layers of liquid, enabling faster diffusion so that even more slowly diffusing molecules, like proteins or nucleic acids, can achieve fast mixing on timescales relevant to biological reactions. This mixer was first used in UV-vis absorbance and SAXS experiments with systems of a variety of molecular weights, and thus diffusion speeds. Careful effort was also dedicated to making a loop-loading sample-delivery system that consumes as little sample as possible, enabling the study of precious, laboratory-purified samples. The combination of the versatile mixer with low sample consumption opens the door to many new applications for mix-and-inject studies.


Assuntos
Microfluídica , Proteínas , Difração de Raios X , Espalhamento a Baixo Ângulo , Raios X , Proteínas/química
4.
Methods Enzymol ; 677: 41-83, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36410957

RESUMO

Proteins and nucleic acids, alone and in complex are among the essential building blocks of living organisms. Obtaining a molecular level understanding of their structures, and the changes that occur as they interact, is critical for expanding our knowledge of life processes or disease progression. Here, we motivate and describe an application of solution small angle X-ray scattering (SAXS) which provides valuable information about the structures, ensembles, compositions and dynamics of protein-nucleic acid complexes in solution, in equilibrium and time-resolved studies. Contrast variation (CV-) SAXS permits the visualization of the distinct molecular constituents (protein and/or nucleic acid) within a complex. CV-SAXS can be implemented in two modes. In the simplest, the protein within the complex is effectively rendered invisible by the addition of an inert contrast agent at an appropriate concentration. Under these conditions, the structure, or structural changes of only the nucleic acid component of the complex can be studied in detail. The second mode permits observation of both components of the complex: the protein and the nucleic acid. This approach requires the acquisition of SAXS profiles on the complex at different concentrations of a contrast agent. Here, we review CV-SAXS as applied to protein-nucleic acid complexes in both modes. We provide some theoretical framework for CV-SAXS but focus primarily on providing the necessary information required to implement a successful experiment including experimental design, sample quality assessment, and data analysis.


Assuntos
Análise de Dados , Ácidos Nucleicos , Espalhamento a Baixo Ângulo , Difração de Raios X , Projetos de Pesquisa , Meios de Contraste , Proteínas/química , Literatura de Revisão como Assunto
5.
Proc Natl Acad Sci U S A ; 118(14)2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33785601

RESUMO

Cis-acting RNA elements are crucial for the regulation of polyadenylated RNA stability. The element for nuclear expression (ENE) contains a U-rich internal loop flanked by short helices. An ENE stabilizes RNA by sequestering the poly(A) tail via formation of a triplex structure that inhibits a rapid deadenylation-dependent decay pathway. Structure-based bioinformatic studies identified numerous ENE-like elements in evolutionarily diverse genomes, including a subclass containing two ENE motifs separated by a short double-helical region (double ENEs [dENEs]). Here, the structure of a dENE derived from a rice transposable element (TWIFB1) before and after poly(A) binding (∼24 kDa and ∼33 kDa, respectively) is investigated. We combine biochemical structure probing, small angle X-ray scattering (SAXS), and cryo-electron microscopy (cryo-EM) to investigate the dENE structure and its local and global structural changes upon poly(A) binding. Our data reveal 1) the directionality of poly(A) binding to the dENE, and 2) that the dENE-poly(A) interaction involves a motif that protects the 3'-most seven adenylates of the poly(A). Furthermore, we demonstrate that the dENE does not undergo a dramatic global conformational change upon poly(A) binding. These findings are consistent with the recently solved crystal structure of a dENE+poly(A) complex [S.-F. Torabi et al., Science 371, eabe6523 (2021)]. Identification of additional modes of poly(A)-RNA interaction opens new venues for better understanding of poly(A) tail biology.


Assuntos
Poliadenilação , Estabilidade de RNA , RNA/química , Elementos de DNA Transponíveis , Células HEK293 , Humanos , Motivos de Nucleotídeos , Oryza/genética , RNA/metabolismo
6.
Biophys J ; 119(12): 2524-2536, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33189689

RESUMO

Although conformational dynamics of RNA molecules are potentially important in microRNA (miRNA) processing, the role of the protein binding partners in facilitating the requisite structural changes is not well understood. In previous work, we and others have demonstrated that nonduplex structural elements and the conformational flexibility they support are necessary for efficient RNA binding and cleavage by the proteins associated with the two major stages of miRNA processing. However, recent studies showed that the protein DGCR8 binds primary miRNA and duplex RNA with similar affinities. Here, we study RNA binding by a small recombinant construct of the DGCR8 protein and the RNA conformation changes that result. This construct, the DGCR8 core, contains two double-stranded RNA-binding domains (dsRBDs) and a C-terminal tail. To assess conformational changes resulting from binding, we applied small-angle x-ray scattering with contrast variation to detect conformational changes of primary-miR-16-1 in complex with the DGCR8 core. This method reports only on the RNA conformation within the complex and suggests that the protein bends the RNA upon binding. Supporting work using smFRET to study the conformation of RNA duplexes bound to the core also shows bending. Together, these studies elucidate the role of DGCR8 in interacting with RNA during the early stages of miRNA processing.


Assuntos
MicroRNAs , Proteínas de Ligação a RNA , MicroRNAs/genética , MicroRNAs/metabolismo , Microcomputadores , Conformação de Ácido Nucleico , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo
7.
RNA ; 24(12): 1828-1838, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30254137

RESUMO

Folding of an RNA from secondary to tertiary structure often depends on divalent ions for efficient electrostatic charge screening (nonspecific association) or binding (specific association). To measure how different divalent cations modify folding kinetics of the 60 nucleotide Ecoli rRNA GTPase center, we combined stopped-flow fluorescence in the presence of Mg2+, Ca2+, or Sr2+ together with time-resolved small angle X-ray scattering (SAXS) in the presence of Mg2+ to observe the folding process. Immediately upon addition of each divalent ion, the RNA undergoes a transition from an extended state with secondary structure to a more compact structure. Subsequently, specific divalent ions modulate populations of intermediates in conformational ensembles along the folding pathway with transition times longer than 10 msec. Rate constants for the five folding transitions act on timescales from submillisecond to tens of seconds. The sensitivity of RNA tertiary structure to divalent cation identity affects all but the fastest events in RNA folding, and allowed us to identify those states that prefer Mg2+ The GTPase center RNA appears to have optimized its folding trajectory to specifically utilize this most abundant intracellular divalent ion.


Assuntos
GTP Fosfo-Hidrolases/química , Conformação de Ácido Nucleico/efeitos dos fármacos , Dobramento de RNA/efeitos dos fármacos , RNA Ribossômico/química , Cátions Bivalentes/farmacologia , Escherichia coli , Cinética , RNA Ribossômico/genética , Espalhamento a Baixo Ângulo , Difração de Raios X
8.
Biophys J ; 112(1): 22-30, 2017 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-28076812

RESUMO

Interactions between the polyamine spermine and nucleic acids drive important cellular processes. Spermine condenses DNA and some RNAs, such as poly(rA):poly(rU). A large fraction of the spermine present in cells is bound to RNA but apparently does not condense it. Here, we study the effect of spermine binding to short duplex RNA and DNA, and compare our findings with predictions of molecular-dynamics simulations. When small numbers of spermine are introduced, RNA with a designed sequence containing a mixture of 14 GC pairs and 11 AU pairs resists condensation relative to DNA of an equivalent sequence or to 25 bp poly(rA):poly(rU) RNA. A comparison of wide-angle x-ray scattering profiles with simulation results suggests that spermine is sequestered deep within the major groove of mixed-sequence RNA. This prevents condensation by limiting opportunities to bridge to other molecules and stabilizes the RNA by locking it into a particular conformation. In contrast, for DNA, simulations suggest that spermine binds externally to the duplex, offering opportunities for intermolecular interaction. The goal of this study is to explain how RNA can remain soluble and available for interaction with other molecules in the cell despite the presence of spermine at concentrations high enough to precipitate DNA.


Assuntos
DNA/química , Conformação de Ácido Nucleico/efeitos dos fármacos , RNA/química , Espermina/farmacologia , Simulação de Dinâmica Molecular
9.
Proc Natl Acad Sci U S A ; 114(2): 334-339, 2017 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-28028239

RESUMO

The nucleosome core particle (NCP) is the basic structural unit for genome packaging in eukaryotic cells and consists of DNA wound around a core of eight histone proteins. DNA access is modulated through dynamic processes of NCP disassembly. Partly disassembled structures, such as the hexasome (containing six histones) and the tetrasome (four histones), are important for transcription regulation in vivo. However, the pathways for their formation have been difficult to characterize. We combine time-resolved (TR) small-angle X-ray scattering and TR-FRET to correlate changes in the DNA conformations with composition of the histone core during salt-induced disassembly of canonical NCPs. We find that H2A-H2B histone dimers are released sequentially, with the first dimer being released after the DNA has formed an asymmetrically unwrapped, teardrop-shape DNA structure. This finding suggests that the octasome-to-hexasome transition is guided by the asymmetric unwrapping of the DNA. The link between DNA structure and histone composition suggests a potential mechanism for the action of proteins that alter nucleosome configurations such as histone chaperones and chromatin remodeling complexes.


Assuntos
DNA/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Animais , Cromatina/metabolismo , Conformação de Ácido Nucleico , Xenopus laevis/metabolismo
10.
Biophys Rev ; 8(2): 139-149, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27551324

RESUMO

Understanding how DNA carries out its biological roles requires knowledge of its interactions with biological partners. Since DNA is a polyanionic polymer, electrostatic interactions contribute significantly. These interactions are mediated by positively charged protein residues or charge compensating cations. Direct detection of these partners and/or their effect on DNA conformation poses challenges, especially for monitoring conformational dynamics in real time. Small-angle x-ray scattering (SAXS) is uniquely sensitive to both the conformation and local environment (i.e. protein partner and associated ions) of the DNA. The primary challenge of studying multi-component systems with SAXS lies in resolving how each component contributes to the measured scattering. Here, we review two contrast variation (CV) strategies that enable targeted studies of the structures of DNA or its associated partners. First, solution contrast variation enables measurement of DNA conformation within a protein-DNA complex by masking out the protein contribution to the scattering profile. We review a specific example, in which the real-time unwrapping of DNA from a nucleosome core particle is measured during salt-induced disassembly. The second method, heavy atom isomorphous replacement, reports the spatial distribution of the cation cloud around duplex DNA by exploiting changes in the scattering strength of cations with varying atomic numbers. We demonstrate the application of this approach to provide the spatial distribution of monovalent cations (Na+, K+, Rb+, Cs+) around a standard 25-base pair DNA. The CV strategies presented here are valuable tools for understanding DNA interactions with its biological partners.

11.
J Chem Phys ; 144(21): 214105, 2016 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-27276943

RESUMO

Small-angle X-ray scattering measurements can provide valuable information about the solvent environment around biomolecules, but it can be difficult to extract solvent-specific information from observed intensity profiles. Intensities are proportional to the square of scattering amplitudes, which are complex quantities. Amplitudes in the forward direction are real, and the contribution from a solute of known structure (and from the waters it excludes) can be estimated from theory; hence, the amplitude arising from the solvent environment can be computed by difference. We have found that this "square root subtraction scheme" can be extended to non-zero q values, out to 0.1 Å(-1) for the systems considered here, since the phases arising from the solute and from the water environment are nearly identical in this angle range. This allows us to extract aspects of the water and ion distributions (beyond their total numbers), by combining experimental data for the complete system with calculations for the solutes. We use this approach to test molecular dynamics and integral-equation (3D-RISM (three-dimensional reference interaction site model)) models for solvent structure around myoglobin, lysozyme, and a 25 base-pair duplex DNA. Comparisons can be made both in Fourier space and in terms of the distribution of interatomic distances in real space. Generally, computed solvent distributions arising from the MD simulations fit experimental data better than those from 3D-RISM, even though the total small-angle X-ray scattering patterns are very similar; this illustrates the potential power of this sort of analysis to guide the development of computational models.


Assuntos
Soluções/química , Água/química , DNA/química , Análise de Fourier , Íons/química , Modelos Químicos , Simulação de Dinâmica Molecular , Muramidase/química , Mioglobina/química , Espalhamento a Baixo Ângulo , Difração de Raios X
12.
J Chem Phys ; 144(20): 205102, 2016 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-27250330

RESUMO

Wide-angle x-ray scattering (WAXS) is emerging as a powerful tool for increasing the resolution of solution structure measurements of biomolecules. Compared to its better known complement, small angle x-ray scattering (SAXS), WAXS targets higher scattering angles and can enhance structural studies of molecules by accessing finer details of solution structures. Although the extension from SAXS to WAXS is easy to implement experimentally, the computational tools required to fully harness the power of WAXS are still under development. Currently, WAXS is employed to study structural changes and ligand binding in proteins; however, the methods are not as fully developed for nucleic acids. Here, we show how WAXS can qualitatively characterize nucleic acid structures as well as the small but significant structural changes driven by the addition of multivalent ions. We show the potential of WAXS to test all-atom molecular dynamics (MD) simulations and to provide insight into understanding how the trivalent ion cobalt(III) hexammine (CoHex) affects the structure of RNA and DNA helices. We find that MD simulations capture the RNA structural change that occurs due to addition of CoHex.


Assuntos
Modelos Químicos , Simulação de Dinâmica Molecular , Ácidos Nucleicos/química , Cobalto/química , Conformação de Ácido Nucleico , Espalhamento a Baixo Ângulo , Raios X
13.
Biophys J ; 110(2): 315-326, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26789755

RESUMO

The ionic atmospheres around nucleic acids play important roles in biological function. Large-scale explicit solvent simulations coupled to experimental assays such as anomalous small-angle x-ray scattering can provide important insights into the structure and energetics of such atmospheres but are time- and resource intensive. In this article, we use classical density functional theory to explore the balance among ion-DNA, ion-water, and ion-ion interactions in ionic atmospheres of RbCl, SrCl2, and CoHexCl3 (cobalt hexamine chloride) around a B-form DNA molecule. The accuracy of the classical density functional theory calculations was assessed by comparison between simulated and experimental anomalous small-angle x-ray scattering curves, demonstrating that an accurate model should take into account ion-ion correlation and ion hydration forces, DNA topology, and the discrete distribution of charges on the DNA backbone. As expected, these calculations revealed significant differences among monovalent, divalent, and trivalent cation distributions around DNA. Approximately half of the DNA-bound Rb(+) ions penetrate into the minor groove of the DNA and half adsorb on the DNA backbone. The fraction of cations in the minor groove decreases for the larger Sr(2+) ions and becomes zero for CoHex(3+) ions, which all adsorb on the DNA backbone. The distribution of CoHex(3+) ions is mainly determined by Coulomb and steric interactions, while ion-correlation forces play a central role in the monovalent Rb(+) distribution and a combination of ion-correlation and hydration forces affect the Sr(2+) distribution around DNA. This does not imply that correlations in CoHex solutions are weaker or stronger than for other ions. Steric inaccessibility of the grooves to large CoHex ions leads to their binding at the DNA surface. In this binding mode, first-order electrostatic interactions (Coulomb) dominate the overall binding energy as evidenced by low sensitivity of ionic distribution to the presence or absence of second-order electrostatic correlation interactions.


Assuntos
Cobalto/química , DNA de Forma B/química , Rubídio/química , Estrôncio/química , Eletricidade Estática
14.
Biophys J ; 108(12): 2886-95, 2015 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-26083928

RESUMO

Nucleic acids carry a negative charge, attracting salt ions and water. Interactions with these components of the solvent drive DNA to condense, RNA to fold, and proteins to bind. To understand these biological processes, knowledge of solvent structure around the nucleic acids is critical. Yet, because they are often disordered, ions and water evade detection by x-ray crystallography and other high-resolution methods. Small-angle x-ray scattering (SAXS) is uniquely sensitive to the spatial correlations between solutes and the surrounding solvent. Thus, SAXS provides an experimental constraint to guide or test emerging solvation theories. However, the interpretation of SAXS profiles is nontrivial because of the difficulty in separating the scattering signals of each component: the macromolecule, ions, and hydration water. Here, we demonstrate methods for robustly deconvoluting these signals, facilitating a more straightforward comparison with theory. Using SAXS data collected on an absolute intensity scale for short DNA duplexes in solution with Na(+), K(+), Rb(+), or Cs(+) counterions, we mathematically decompose the scattering profiles into components (DNA, water, and ions) and validate the decomposition using anomalous scattering measurements. In addition, we generate a library of physically motivated ion atmosphere models and rank them by agreement with the scattering data. The best-fit models have relatively compact ion atmospheres when compared to predictions from the mean-field Poisson-Boltzmann theory of electrostatics. Thus, the x-ray scattering methods presented here provide a valuable measurement of the global structure of the ion atmosphere that can be used to test electrostatics theories that go beyond the mean-field approximation.


Assuntos
DNA/química , Metais Alcalinos/química , Simulação de Dinâmica Molecular , Sequência de Bases , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Espalhamento a Baixo Ângulo , Água/química , Difração de Raios X
15.
J Chem Phys ; 141(22): 22D508, 2014 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-25494779

RESUMO

A new method is introduced to compute X-ray solution scattering profiles from atomic models of macromolecules. The three-dimensional version of the Reference Interaction Site Model (RISM) from liquid-state statistical mechanics is employed to compute the solvent distribution around the solute, including both water and ions. X-ray scattering profiles are computed from this distribution together with the solute geometry. We describe an efficient procedure for performing this calculation employing a Lebedev grid for the angular averaging. The intensity profiles (which involve no adjustable parameters) match experiment and molecular dynamics simulations up to wide angle for two proteins (lysozyme and myoglobin) in water, as well as the small-angle profiles for a dozen biomolecules taken from the BioIsis.net database. The RISM model is especially well-suited for studies of nucleic acids in salt solution. Use of fiber-diffraction models for the structure of duplex DNA in solution yields close agreement with the observed scattering profiles in both the small and wide angle scattering (SAXS and WAXS) regimes. In addition, computed profiles of anomalous SAXS signals (for Rb(+) and Sr(2+)) emphasize the ionic contribution to scattering and are in reasonable agreement with experiment. In cases where an absolute calibration of the experimental data at q = 0 is available, one can extract a count of the excess number of waters and ions; computed values depend on the closure that is assumed in the solution of the Ornstein-Zernike equations, with results from the Kovalenko-Hirata closure being closest to experiment for the cases studied here.


Assuntos
Ácidos Nucleicos/química , Proteínas/química , Água/química , Simulação de Dinâmica Molecular , Muramidase/química , Mioglobina/química , Espalhamento a Baixo Ângulo , Difração de Raios X
16.
Nucleic Acids Res ; 42(16): 10823-31, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25123663

RESUMO

The addition of small amounts of multivalent cations to solutions containing double-stranded DNA leads to inter-DNA attraction and eventual condensation. Surprisingly, the condensation is suppressed in double-stranded RNA, which carries the same negative charge as DNA, but assumes a different double helical form. Here, we combine experiment and atomistic simulations to propose a mechanism that explains the variations in condensation of short (25 base-pairs) nucleic acid (NA) duplexes, from B-like form of homopolymeric DNA, to mixed sequence DNA, to DNA:RNA hybrid, to A-like RNA. Circular dichroism measurements suggest that duplex helical geometry is not the fundamental property that ultimately determines the observed differences in condensation. Instead, these differences are governed by the spatial variation of cobalt hexammine (CoHex) binding to NA. There are two major NA-CoHex binding modes--internal and external--distinguished by the proximity of bound CoHex to the helical axis. We find a significant difference, up to 5-fold, in the fraction of ions bound to the external surfaces of the different NA constructs studied. NA condensation propensity is determined by the fraction of CoHex ions in the external binding mode.


Assuntos
RNA de Cadeia Dupla/química , Cobalto/metabolismo , DNA de Forma B/química , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , RNA de Cadeia Dupla/metabolismo , Eletricidade Estática
17.
Nucleic Acids Res ; 42(13): 8767-76, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24990379

RESUMO

The modulation of DNA accessibility by nucleosomes is a fundamental mechanism of gene regulation in eukaryotes. The nucleosome core particle (NCP) consists of 147 bp of DNA wrapped around a symmetric octamer of histone proteins. The dynamics of DNA packaging and unpackaging from the NCP affect all DNA-based chemistries, but depend on many factors, including DNA positioning sequence, histone variants and modifications. Although the structure of the intact NCP has been studied by crystallography at atomic resolution, little is known about the structures of the partially unwrapped, transient intermediates relevant to nucleosome dynamics in processes such as transcription, DNA replication and repair. We apply a new experimental approach combining contrast variation with time-resolved small angle X-ray scattering (TR-SAXS) to determine transient structures of protein and DNA constituents of NCPs during salt-induced disassembly. We measure the structures of unwrapping DNA and monitor protein dissociation from Xenopus laevis histones reconstituted with two model NCP positioning constructs: the Widom 601 sequence and the sea urchin 5S ribosomal gene. Both constructs reveal asymmetric release of DNA from disrupted histone cores, but display different patterns of protein dissociation. These kinetic intermediates may be biologically important substrates for gene regulation.


Assuntos
DNA/química , Nucleossomos/química , Conformação de Ácido Nucleico , Espalhamento a Baixo Ângulo , Cloreto de Sódio/química , Difração de Raios X
18.
Biopolymers ; 99(12): 1032-45, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23606337

RESUMO

Nucleic acids are highly charged polyelectrolytes that interact strongly with salt ions. Rigid, base-paired regions are successfully described with wormlike chain models, but nonbase-paired single stranded regions have fundamentally different polymer properties because of their greater flexibility. Recently, attention has turned to single stranded nucleic acids due to the growing recognition of their biological importance, as well as the availability of sophisticated experimental techniques sensitive to the conformation of individual molecules. We investigate polyelectrolyte properties of poly(dT), an important and widely studied model system for flexible single stranded nucleic acids, in physiologically important mixed mono- and divalent salt. We report measurements of the form factor and interparticle interactions using SAXS, end-to-end distances using smFRET, and number of excess ions using ASAXS. We present a coarse-grained model that accounts for flexibility, excluded volume, and electrostatic interactions in these systems. Predictions of the model are validated against experiment. We also discuss the state of all-atom, explicit solvent molecular dynamics simulations of poly(dT), the next step in understanding the complexities of ion interactions with these highly charged and flexible polymers.


Assuntos
DNA de Cadeia Simples , Espalhamento a Baixo Ângulo , DNA , Íons , Conformação de Ácido Nucleico , Eletricidade Estática , Difração de Raios X
19.
Biochemistry ; 52(9): 1539-46, 2013 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-23398396

RESUMO

Following the addition of ions to trigger folding, RNA molecules undergo a transition from rigid, extended states to a compact ensemble. Determining the time scale for this collapse provides important insights into electrostatic contributions to RNA folding; however, it can be challenging to isolate the effects of purely nonspecific collapse, e.g., relaxation due to backbone charge compensation, from the concurrent formation of some tertiary contacts. To solve this problem, we decoupled nonspecific collapse from tertiary folding using a single-point mutation to eliminate tertiary contacts in the small RNA subdomain known as tP5abc. Microfluidic mixing with microsecond time resolution and Förster resonance energy transfer detection provides insight into the ionic strength-dependent transition from extended to compact ensembles. Differences in reaction rates are detected when folding is initiated by monovalent or divalent ions, consistent with equilibrium measurements illustrating the enhanced screening of divalent ions relative to monovalent ions at the same ionic strength. Ion-driven collapse is fast, and a comparison of the collapse time of the wild-type and mutant tP5abc suggests that site binding of Mg(2+) occurs on submillisecond time scales.


Assuntos
Íons/metabolismo , RNA Catalítico/química , RNA Catalítico/metabolismo , Tetrahymena/enzimologia , Sequência de Bases , Transferência Ressonante de Energia de Fluorescência , Íons/química , Modelos Moleculares , Conformação de Ácido Nucleico , Concentração Osmolar , Mutação Puntual , RNA Catalítico/genética , Tetrahymena/química , Tetrahymena/genética , Tetrahymena/metabolismo
20.
Biophys J ; 102(4): 819-28, 2012 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-22385853

RESUMO

RNA molecules play critical roles in many cellular processes. Traditionally viewed as genetic messengers, RNA molecules were recently discovered to have diverse functions related to gene regulation and expression. RNA also has great potential as a therapeutic and a tool for further investigation of gene regulation. Metal ions are an integral part of RNA structure and should be considered in any experimental or theoretical study of RNA. Here, we report a multidisciplinary approach that combines anomalous small-angle x-ray scattering and molecular-dynamics (MD) simulations with explicit solvent and ions around RNA. From experiment and simulation results, we find excellent agreement in the number and distribution of excess monovalent and divalent ions around a short RNA duplex. Although similar agreement can be obtained from a continuum description of the solvent and mobile ions (by solving the Poisson-Boltzmann equation and accounting for finite ion size), the use of MD is easily extended to flexible RNA systems with thermal fluctuations. Therefore, we also model a short RNA pseudoknot and find good agreement between the MD results and the experimentally derived solution structures. Surprisingly, both deviate from crystal structure predictions. These favorable comparisons of experiment and simulations encourage work on RNA in all-atom dynamic models.


Assuntos
Simulação de Dinâmica Molecular , RNA/química , Espalhamento a Baixo Ângulo , Difração de Raios X , Sequência de Bases , Íons/química , Conformação de Ácido Nucleico , RNA/genética , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , Soluções , Água/química
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