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1.
Mol Cell ; 80(1): 127-139.e6, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007253

RESUMO

Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4 Å core resolution and 4.5-5.7 Å at its periphery, and aided by protein crosslinking we determine its molecular architecture. Our structure provides additional insights into the spliceosome's architecture between the catalytic steps of splicing, and how proteins aid formation of the spliceosome's catalytically active RNP (ribonucleoprotein) conformation. It reveals the spatial organization of the metazoan-specific proteins PPWD1, WDR70, FRG1, and CIR1 in human C complexes, indicating they stabilize functionally important protein domains and RNA structures rearranged/repositioned during the Bact to C transition. Structural comparisons with human Bact, C∗, and P complexes reveal an intricate cascade of RNP rearrangements during splicing catalysis, with intermediate RNP conformations not found in yeast, and additionally elucidate the structural basis for the sequential recruitment of metazoan-specific spliceosomal proteins.


Assuntos
Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Spliceossomos/metabolismo , Animais , Catálise , Células HeLa , Humanos , Íntrons/genética , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Estabilidade Proteica , RNA/química , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Fatores de Tempo
2.
Phys Rev Lett ; 125(7): 078003, 2020 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-32857587

RESUMO

Determining the positions of lattice defects on bounded elastic surfaces with Gaussian curvature is a nontrivial task of mechanical energy optimization. We introduce a simple way to predict the onset of disclination disorder from the shape of the surface. The criterion fixes the value of a weighted integral Gaussian curvature to a universal constant and proves accurate across a great variety of shapes. It provides improved understanding of the limitations to crystalline order in many natural and engineering contexts, such as the assembly of viral capsids.


Assuntos
Capsídeo/química , Modelos Teóricos , RNA/química , Animais , Proteínas do Capsídeo/química , Drosophila , Elasticidade , HIV/química , Vírus da SARS/química , Propriedades de Superfície , Termodinâmica , Thermoproteus
3.
Phys Rev Lett ; 125(4): 048104, 2020 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-32794805

RESUMO

The RNA world scenario posits replication by RNA polymerases. On early Earth, a geophysical setting is required to separate hybridized strands after their replication and to localize them against diffusion. We present a pointed heat source that drives exponential, RNA-catalyzed amplification of short RNA with high efficiency in a confined chamber. While shorter strands were periodically melted by laminar convection, the temperature gradient caused aggregated polymerase molecules to accumulate, protecting them from degradation in hot regions of the chamber. These findings demonstrate a size-selective pathway for autonomous RNA-based replication in natural nonequilibrium conditions.


Assuntos
Ecossistema , RNA/química , RNA/genética , Catálise , DNA/química , DNA/genética , DNA/metabolismo , Replicação do DNA , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Planeta Terra , Evolução Molecular , Temperatura Alta , Biossíntese de Proteínas/genética , RNA/metabolismo
4.
BMC Bioinformatics ; 21(1): 375, 2020 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859148

RESUMO

BACKGROUND: As the barriers to incorporating RNA sequencing (RNA-Seq) into biomedical studies continue to decrease, the complexity and size of RNA-Seq experiments are rapidly growing. Paired, longitudinal, and other correlated designs are becoming commonplace, and these studies offer immense potential for understanding how transcriptional changes within an individual over time differ depending on treatment or environmental conditions. While several methods have been proposed for dealing with repeated measures within RNA-Seq analyses, they are either restricted to handling only paired measurements, can only test for differences between two groups, and/or have issues with maintaining nominal false positive and false discovery rates. In this work, we propose a Bayesian hierarchical negative binomial generalized linear mixed model framework that can flexibly model RNA-Seq counts from studies with arbitrarily many repeated observations, can include covariates, and also maintains nominal false positive and false discovery rates in its posterior inference. RESULTS: In simulation studies, we showed that our proposed method (MCMSeq) best combines high statistical power (i.e. sensitivity or recall) with maintenance of nominal false positive and false discovery rates compared the other available strategies, especially at the smaller sample sizes investigated. This behavior was then replicated in an application to real RNA-Seq data where MCMSeq was able to find previously reported genes associated with tuberculosis infection in a cohort with longitudinal measurements. CONCLUSIONS: Failing to account for repeated measurements when analyzing RNA-Seq experiments can result in significantly inflated false positive and false discovery rates. Of the methods we investigated, whether they model RNA-Seq counts directly or worked on transformed values, the Bayesian hierarchical model implemented in the mcmseq R package (available at https://github.com/stop-pre16/mcmseq ) best combined sensitivity and nominal error rate control.


Assuntos
RNA/química , Análise de Sequência de RNA/métodos , Interface Usuário-Computador , Teorema de Bayes , Humanos , Método de Monte Carlo , RNA/genética , RNA/metabolismo , Tuberculose/genética , Tuberculose/patologia
5.
PLoS One ; 15(8): e0237747, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822369

RESUMO

With the great significance of biomolecular flexibility in biomolecular dynamics and functional analysis, various experimental and theoretical models are developed. Experimentally, Debye-Waller factor, also known as B-factor, measures atomic mean-square displacement and is usually considered as an important measurement for flexibility. Theoretically, elastic network models, Gaussian network model, flexibility-rigidity model, and other computational models have been proposed for flexibility analysis by shedding light on the biomolecular inner topological structures. Recently, a topology-based machine learning model has been proposed. By using the features from persistent homology, this model achieves a remarkable high Pearson correlation coefficient (PCC) in protein B-factor prediction. Motivated by its success, we propose weighted-persistent-homology (WPH)-based machine learning (WPHML) models for RNA flexibility analysis. Our WPH is a newly-proposed model, which incorporate physical, chemical and biological information into topological measurements using a weight function. In particular, we use local persistent homology (LPH) to focus on the topological information of local regions. Our WPHML model is validated on a well-established RNA dataset, and numerical experiments show that our model can achieve a PCC of up to 0.5822. The comparison with the previous sequence-information-based learning models shows that a consistent improvement in performance by at least 10% is achieved in our current model.


Assuntos
RNA/química , Algoritmos , Elasticidade , Aprendizado de Máquina , Distribuição Normal , Conformação de Ácido Nucleico
6.
Nat Commun ; 11(1): 3422, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647223

RESUMO

The Integrator complex processes 3'-ends of spliceosomal small nuclear RNAs (snRNAs). Furthermore, it regulates transcription of protein coding genes by terminating transcription after unstable pausing. The molecular basis for Integrator's functions remains obscure. Here, we show that INTS10, Asunder/INTS13 and INTS14 form a separable, functional Integrator module. The structure of INTS13-INTS14 reveals a strongly entwined complex with a unique chain interlink. Unexpected structural homology to the Ku70-Ku80 DNA repair complex suggests nucleic acid affinity. Indeed, the module displays affinity for DNA and RNA but prefers RNA hairpins. While the module plays an accessory role in snRNA maturation, it has a stronger influence on transcription termination after pausing. Asunder/INTS13 directly binds Integrator's cleavage module via a conserved C-terminal motif that is involved in snRNA processing and required for spermatogenesis. Collectively, our data establish INTS10-INTS13-INTS14 as a nucleic acid-binding module and suggest that it brings cleavage module and target transcripts into proximity.


Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ácidos Nucleicos/metabolismo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Autoantígeno Ku/química , Mutação/genética , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , RNA/química , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Homologia Estrutural de Proteína
7.
Nucleic Acids Res ; 48(13): 7520-7531, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32678884

RESUMO

2'-5'-Oligoadenylate synthetases (OAS) are innate immune sensors of cytosolic double-stranded RNA (dsRNA) and play a critical role in limiting viral infection. dsRNA binding induces allosteric structural changes in OAS1 that reorganize its catalytic center to promote synthesis of 2'-5'-oligoadenylate and thus activation of endoribonuclease L. Specific RNA sequences and structural motifs can also enhance activation of OAS1 through currently undefined mechanisms. To better understand these drivers of OAS activation, we tested the impact of defined sequence changes within a short dsRNA that strongly activates OAS1. Both in vitro and in human A549 cells, appending a 3'-end single-stranded pyrimidine (3'-ssPy) can strongly enhance OAS1 activation or have no effect depending on its location, suggesting that other dsRNA features are necessary for correct presentation of the motif to OAS1. Consistent with this idea, we also find that the dsRNA binding position is dictated by an established consensus sequence (WWN9WG). Unexpectedly, however, not all sequences fitting this consensus activate OAS1 equivalently, with strong dependence on the identity of both partially conserved (W) and non-conserved (N9) residues. A picture thus emerges in which both specific RNA features and the context in which they are presented dictate the ability of short dsRNAs to activate OAS1.


Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Sequência Consenso , RNA/química , 2',5'-Oligoadenilato Sintetase/química , Células A549 , Regulação Alostérica , Sítio Alostérico , Domínio Catalítico , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , RNA/metabolismo
8.
Postepy Biochem ; 66(2): 111-124, 2020 06 27.
Artigo em Polonês | MEDLINE | ID: mdl-32700504

RESUMO

Membraneless organelles (MLOs) are a large group of intracellular compartments formed during various stages of a cell life. They are important subcellular structures which enable a cell performance of vital physiological processes including stress response. MLOs can be found in cytoplasm and organelles that are sealed by lipidic membrane, mainly in nucleus. They are formed by the thermodynamically driven liquid-liquid phase separation (LLPS). MLOs contain proteins possessing intrinsically disordered regions (IDRs) which together with RNA spontaneously phase separate from the surrounding milieu. This paper presents information on the biophysical basses of the formation and functionality of MLOs. It also discusses a range of experimental techniques that can be applied in biochemical and biological studies of these sub-cellular structures.


Assuntos
Organelas/química , Núcleo Celular , Citoplasma , Proteínas/química , RNA/química
9.
Nat Commun ; 11(1): 3392, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636376

RESUMO

G-quadruplex (G4) is a noncanonical secondary structure of DNA or RNA which can enhance or repress gene expression, yet the underlying molecular mechanism remains uncertain. Here we show that when positioned downstream of transcription start site, the orientation of potential G4 forming sequence (PQS), but not the sequence alters transcriptional output. Ensemble in vitro transcription assays indicate that PQS in the non-template increases mRNA production rate and yield. Using sequential single molecule detection stages, we demonstrate that while binding and initiation of T7 RNA polymerase is unchanged, the efficiency of elongation and the final mRNA output is higher when PQS is in the non-template. Strikingly, the enhanced elongation arises from the transcription-induced R-loop formation, which in turn generates G4 structure in the non-template. The G4 stabilized R-loop leads to increased transcription by a mechanism involving successive rounds of R-loop formation.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Quadruplex G , Estruturas R-Loop , Transcrição Genética , Proteínas Virais/genética , DNA/análise , DNA/química , RNA Polimerases Dirigidas por DNA/química , Transferência Ressonante de Energia de Fluorescência , Ligação Proteica , RNA/química , RNA Mensageiro/química , Sítio de Iniciação de Transcrição , Proteínas Virais/química
10.
Theranostics ; 10(16): 7150-7162, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32641984

RESUMO

In December 2019, a new coronavirus disease (COVID-19) outbreak occurred in Wuhan, China. Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), which is the seventh coronavirus known to infect humans, is highly contagious and has rapidly expanded worldwide since its discovery. Quantitative nucleic acid testing has become the gold standard for diagnosis and guiding clinical decisions regarding the use of antiviral therapy. However, the RT-qPCR assays targeting SARS-CoV-2 have a number of challenges, especially in terms of primer design. Primers are the pivotal components of a RT-qPCR assay. Once virus mutation and recombination occur, it is difficult to effectively diagnose viral infection by existing RT-qPCR primers. Some primers and probes have also been made available on the WHO website for reference. However, no previous review has systematically compared the previously reported primers and probes and described how to design new primers in the event of a new coronavirus infection. This review focuses on how primers and probes can be designed methodically and rationally, and how the sensitivity and specificity of the detection process can be improved. This brief review will be useful for the accurate diagnosis and timely treatment of the new coronavirus pneumonia.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , RNA Viral/genética , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sequência de Bases , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Desenho de Fármacos , Genes Virais , Humanos , Conformação de Ácido Nucleico , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , RNA/química , Sondas RNA/genética , RNA Viral/química , Reação em Cadeia da Polimerase em Tempo Real/estatística & dados numéricos , Sensibilidade e Especificidade , Nanomedicina Teranóstica
11.
Nucleic Acids Res ; 48(14): 7623-7639, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32644123

RESUMO

RNA therapeutics are a promising strategy to treat genetic diseases caused by the overexpression or aberrant splicing of a specific protein. The field has seen major strides in the clinical efficacy of this class of molecules, largely due to chemical modifications and delivery strategies that improve nuclease resistance and enhance cell penetration. However, a major obstacle in the development of RNA therapeutics continues to be the imprecise, difficult, and often problematic nature of most methods used to measure cell penetration. Here, we review these methods and clearly distinguish between those that measure total cellular uptake of RNA therapeutics, which includes both productive and non-productive uptake, and those that measure cytosolic/nuclear penetration, which represents only productive uptake. We critically analyze the benefits and drawbacks of each method. Finally, we use key examples to illustrate how, despite rigorous experimentation and proper controls, our understanding of the mechanism of gymnotic uptake of RNA therapeutics remains limited by the methods commonly used to analyze RNA delivery.


Assuntos
RNA/metabolismo , RNA/uso terapêutico , Aptâmeros de Nucleotídeos/uso terapêutico , Núcleo Celular/metabolismo , Citosol/metabolismo , Doenças Genéticas Inatas/tratamento farmacológico , Técnicas Genéticas , Humanos , MicroRNAs/uso terapêutico , Microscopia Eletrônica , Oligonucleotídeos Antissenso/uso terapêutico , RNA/química , RNA/farmacocinética , RNA Interferente Pequeno/uso terapêutico , Espectrometria de Fluorescência
12.
Nucleic Acids Res ; 48(14): 7690-7699, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32652015

RESUMO

RNA-small molecule binding is a key regulatory mechanism which can stabilize 3D structures and activate molecular functions. The discovery of RNA-targeting compounds is thus a current topic of interest for novel therapies. Our work is a first attempt at bringing the scalability and generalization abilities of machine learning methods to the problem of RNA drug discovery, as well as a step towards understanding the interactions which drive binding specificity. Our tool, RNAmigos, builds and encodes a network representation of RNA structures to predict likely ligands for novel binding sites. We subject ligand predictions to virtual screening and show that we are able to place the true ligand in the 71st-73rd percentile in two decoy libraries, showing a significant improvement over several baselines, and a state of the art method. Furthermore, we observe that augmenting structural networks with non-canonical base pairing data is the only representation able to uncover a significant signal, suggesting that such interactions are a necessary source of binding specificity. We also find that pre-training with an auxiliary graph representation learning task significantly boosts performance of ligand prediction. This finding can serve as a general principle for RNA structure-function prediction when data is scarce. RNAmigos shows that RNA binding data contains structural patterns with potential for drug discovery, and provides methodological insights for possible applications to other structure-function learning tasks. The source code, data and a Web server are freely available at http://rnamigos.cs.mcgill.ca.


Assuntos
RNA/química , Software , Pareamento de Bases , Sítios de Ligação , Ligantes , Conformação de Ácido Nucleico
13.
Nat Methods ; 17(7): 699-707, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32616928

RESUMO

The discovery and design of biologically important RNA molecules is outpacing three-dimensional structural characterization. Here, we demonstrate that cryo-electron microscopy can routinely resolve maps of RNA-only systems and that these maps enable subnanometer-resolution coordinate estimation when complemented with multidimensional chemical mapping and Rosetta DRRAFTER computational modeling. This hybrid 'Ribosolve' pipeline detects and falsifies homologies and conformational rearrangements in 11 previously unknown 119- to 338-nucleotide protein-free RNA structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length Vibrio cholerae and Fusobacterium nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and the computer-designed ATP-TTR-3 aptamer with and without AMP. Simulation benchmarks, blind challenges, compensatory mutagenesis, cross-RNA homologies and internal controls demonstrate that Ribosolve can accurately resolve the global architectures of RNA molecules but does not resolve atomic details. These tests offer guidelines for making inferences in future RNA structural studies with similarly accelerated throughput.


Assuntos
Microscopia Crioeletrônica/métodos , RNA/química , Simulação por Computador , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Catalítico/química , Riboswitch
14.
Nucleic Acids Res ; 48(15): 8675-8685, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32687167

RESUMO

Along with nucleobase pairing, base-base stacking interactions are one of the two main types of strong non-covalent interactions that define the unique secondary and tertiary structure of RNA. In this paper we studied two subfamilies of nucleobase-inserted stacking structures: (i) with any base intercalated between neighboring nucleotide residues (base-intercalated element, BIE, i + 1); (ii) with any base wedged into a hydrophobic cavity formed by heterocyclic bases of two nucleotides which are one nucleotide apart in sequence (base-wedged element, BWE, i + 2). We have exploited the growing database of natively folded RNA structures in Protein Data Bank to analyze the distribution and structural role of these motifs in RNA. We found that these structural elements initially found in yeast tRNAPhe are quite widespread among the tertiary structures of various RNAs. These motifs perform diverse roles in RNA 3D structure formation and its maintenance. They contribute to the folding of RNA bulges and loops and participate in long-range interactions of single-stranded stretches within RNA macromolecules. Furthermore, both base-intercalated and base-wedged motifs participate directly or indirectly in the formation of RNA functional centers, which interact with various ligands, antibiotics and proteins.


Assuntos
Complexos Multiproteicos/ultraestrutura , Conformação de Ácido Nucleico , Proteínas de Ligação a RNA/ultraestrutura , RNA/ultraestrutura , Antibacterianos/química , Pareamento de Bases/genética , Substâncias Intercalantes/química , Ligantes , Modelos Moleculares , Conformação Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Nucleotídeos/química , Nucleotídeos/genética , RNA/química , RNA/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
15.
Proc Natl Acad Sci U S A ; 117(25): 14194-14201, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32522884

RESUMO

The intracellular environment is crowded and heterogeneous. Although the thermodynamic stability of nucleic acid duplexes is predictable in dilute solutions, methods of predicting such stability under specific intracellular conditions are not yet available. We recently showed that the nearest-neighbor model for self-complementary DNA is valid under molecular crowding condition of 40% polyethylene glycol with an average molecular weight of 200 (PEG 200) in 100 mM NaCl. Here, we determined nearest-neighbor parameters for DNA duplex formation under the same crowding condition to predict the thermodynamics of DNA duplexes in the intracellular environment. Preferential hydration of the nucleotides was found to be the key factor for nearest-neighbor parameters in the crowding condition. The determined parameters were shown to predict the thermodynamic parameters (∆H°, ∆S°, and ∆G°37) and melting temperatures (T m) of the DNA duplexes in the crowding condition with significant accuracy. Moreover, we proposed a general method for predicting the stability of short DNA duplexes in different cosolutes based on the relationship between duplex stability and the water activity of the cosolute solution. The method described herein would be valuable for investigating biological processes that occur under specific intracellular crowded conditions and for the application of DNA-based biotechnologies in crowded environments.


Assuntos
DNA/química , Nucleotídeos/química , Sequência de Bases , DNA/genética , Estrutura Molecular , Conformação de Ácido Nucleico , Polietilenoglicóis , RNA/química , Estabilidade de RNA , Termodinâmica
16.
Nat Commun ; 11(1): 3137, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561731

RESUMO

The close synergy between peptides and nucleic acids in current biology is suggestive of a functional co-evolution between the two polymers. Here we show that cationic proto-peptides (depsipeptides and polyesters), either produced as mixtures from plausibly prebiotic dry-down reactions or synthetically prepared in pure form, can engage in direct interactions with RNA resulting in mutual stabilization. Cationic proto-peptides significantly increase the thermal stability of folded RNA structures. In turn, RNA increases the lifetime of a depsipeptide by >30-fold. Proto-peptides containing the proteinaceous amino acids Lys, Arg, or His adjacent to backbone ester bonds generally promote RNA duplex thermal stability to a greater magnitude than do analogous sequences containing non-proteinaceous residues. Our findings support a model in which tightly-intertwined biological dependencies of RNA and protein reflect a long co-evolutionary history that began with rudimentary, mutually-stabilizing interactions at early stages of polypeptide and nucleic acid co-existence.


Assuntos
Evolução Molecular , Peptídeos/metabolismo , Dobramento de Proteína , Estabilidade de RNA , RNA/metabolismo , Sequência de Aminoácidos , Aminobutiratos/química , Aminobutiratos/metabolismo , Cátions/química , Cátions/metabolismo , Dicroísmo Circular , Hidrólise , Ressonância Magnética Nuclear Biomolecular , Origem da Vida , Ornitina/química , Ornitina/metabolismo , Peptídeos/química , Estabilidade Proteica , RNA/química , beta-Alanina/análogos & derivados , beta-Alanina/química , beta-Alanina/metabolismo
17.
Nat Commun ; 11(1): 2946, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32522989

RESUMO

Aptamer switches that respond sensitively to pH could enhance control over molecular devices, improving their diagnostic and therapeutic efficacy. Previous designs have inserted pH-sensitive DNA motifs into aptamer sequences. Unfortunately, their performance was limited by the motifs' intrinsic pH-responses and could not be tuned to operate across arbitrary pH ranges. Here, we present a methodology for converting virtually any aptamer into a molecular switch with pH-selective binding properties - in acidic, neutral, or alkaline conditions. Our design inserts two orthogonal motifs that can be manipulated in parallel to tune pH-sensitivity without altering the aptamer sequence itself. From a single ATP aptamer, we engineer pH-controlled target binding under diverse conditions, achieving pH-induced selectivity in affinity of up to 1,000-fold. Importantly, we demonstrate the design of tightly regulated aptamers with strong target affinity over only a narrow pH range. Our approach offers a highly generalizable strategy for integrating pH-responsiveness into molecular devices.


Assuntos
Técnicas Biossensoriais , DNA/química , Concentração de Íons de Hidrogênio , Nanotecnologia/métodos , RNA/química , Biologia Sintética
18.
Nucleic Acids Res ; 48(13): 7502-7519, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32542384

RESUMO

The regulation of gene expression by small RNAs in Escherichia coli depends on RNA binding proteins Hfq and ProQ, which bind mostly distinct RNA pools. To understand how ProQ discriminates between RNA substrates, we compared its binding to six different RNA molecules. Full-length ProQ bound all six RNAs similarly, while the isolated N-terminal FinO domain (NTD) of ProQ specifically recognized RNAs with Rho-independent terminators. Analysis of malM 3'-UTR mutants showed that tight RNA binding by the ProQ NTD required a terminator hairpin of at least 2 bp preceding an 3' oligoU tail of at least four uridine residues. Substitution of an A-rich sequence on the 5' side of the terminator to uridines strengthened the binding of several ProQ-specific RNAs to the Hfq protein, but not to the ProQ NTD. Substitution of the motif in the malM-3' and cspE-3' RNAs also conferred the ability to bind Hfq in E. coli cells, as measured using a three-hybrid assay. In summary, these data suggest that the ProQ NTD specifically recognizes 3' intrinsic terminators of RNA substrates, and that the discrimination between RNA ligands by E. coli ProQ and Hfq depends both on positive determinants for binding to ProQ and negative determinants against binding to Hfq.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Ligação a RNA/química , Sítios de Ligação , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/química , Fator Proteico 1 do Hospedeiro/metabolismo , Mutação , Motivos de Nucleotídeos , Ligação Proteica , RNA/química , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
19.
Nucleic Acids Res ; 48(12): 6503-6512, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32491164

RESUMO

An RNA global fold can be described at the level of helix orientations and relatively flexible loop conformations that connect the helices. The linkage between the helices plays an essential role in determining the structural topology, which restricts RNA local and global folds, especially for RNA tertiary structures involving cross-linked base pairs. We quantitatively analyze the topological constraints on RNA 3D conformational space, in particular, on the distribution of helix orientations, for pseudoknots and loop-loop kissing structures. The result shows that a viable conformational space is predominantly determined by the motif type, helix size, and loop size, indicating a strong topological coupling between helices and loops in RNA tertiary motifs. Moreover, the analysis indicates that (cross-linked) tertiary contacts can cause much stronger topological constraints on RNA global fold than non-cross-linked base pairs. Furthermore, based on the topological constraints encoded in the 2D structure and the 3D templates, we develop a 3D structure prediction approach. This approach can be further combined with structure probing methods to expand the capability of computational prediction for large RNA folds.


Assuntos
Simulação de Dinâmica Molecular , Dobramento de RNA , RNA/química , Motivos de Nucleotídeos
20.
Proc Natl Acad Sci U S A ; 117(27): 15731-15739, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32561643

RESUMO

De novo emergence demands a transition from disordered polypeptides into structured proteins with well-defined functions. However, can polypeptides confer functions of evolutionary relevance, and how might such polypeptides evolve into modern proteins? The earliest proteins present an even greater challenge, as they were likely based on abiotic, spontaneously synthesized amino acids. Here we asked whether a primordial function, such as nucleic acid binding, could emerge with ornithine, a basic amino acid that forms abiotically yet is absent in modern-day proteins. We combined ancestral sequence reconstruction and empiric deconstruction to unravel a gradual evolutionary trajectory leading from a polypeptide to a ubiquitous nucleic acid-binding protein. Intermediates along this trajectory comprise sequence-duplicated functional proteins built from 10 amino acid types, with ornithine as the only basic amino acid. Ornithine side chains were further modified into arginine by an abiotic chemical reaction, improving both structure and function. Along this trajectory, function evolved from phase separation with RNA (coacervates) to avid and specific double-stranded DNA binding. Our results suggest that phase-separating polypeptides may have been an evolutionary resource for the emergence of early proteins, and that ornithine, together with its postsynthesis modification to arginine, could have been the earliest basic amino acids.


Assuntos
Arginina/química , Nucleoproteínas/genética , Ornitina/química , Peptídeos/genética , Sequência de Aminoácidos/genética , Aminoácidos/química , Aminoácidos/genética , Arginina/genética , DNA/química , DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Nucleoproteínas/química , Ornitina/genética , Peptídeos/química , Proteínas/química , Proteínas/genética , RNA/química , RNA/genética
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