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1.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205000

RESUMO

Recently, the 1H-detected in-cell NMR spectroscopy has emerged as a unique tool allowing the characterization of interactions between nucleic acid-based targets and drug-like molecules in living human cells. Here, we assess the application potential of 1H and 19F-detected in-cell NMR spectroscopy to profile drugs/ligands targeting DNA G-quadruplexes, arguably the most studied class of anti-cancer drugs targeting nucleic acids. We show that the extension of the original in-cell NMR approach is not straightforward. The severe signal broadening and overlap of 1H in-cell NMR spectra of polymorphic G-quadruplexes and their complexes complicate their quantitative interpretation. Nevertheless, the 1H in-cell NMR can be used to identify drugs that, despite strong interaction in vitro, lose their ability to bind G-quadruplexes in the native environment. The in-cell NMR approach is adjusted to a recently developed 3,5-bis(trifluoromethyl)phenyl probe to monitor the intracellular interaction with ligands using 19F-detected in-cell NMR. The probe allows dissecting polymorphic mixture in terms of number and relative populations of individual G-quadruplex species, including ligand-bound and unbound forms in vitro and in cellulo. Despite the probe's discussed limitations, the 19F-detected in-cell NMR appears to be a promising strategy to profile G-quadruplex-ligand interactions in the complex environment of living cells.


Assuntos
DNA/efeitos dos fármacos , Quadruplex G/efeitos dos fármacos , Conformação de Ácido Nucleico/efeitos dos fármacos , Preparações Farmacêuticas/química , Sítios de Ligação/efeitos dos fármacos , DNA/química , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Prótons
2.
Molecules ; 26(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34206097

RESUMO

In this contribution, we recall and test a new methodology designed to identify the favorable reaction pathway between two reactants. Applied to the formation of the DNA guanine (G) -cytosine (C) pair, we successfully predict the best orientation between the base pairs held together by hydrogen bonds and leading to the formation of the typical Watson Crick structure of the GC pair. Beyond the global minimum, some local stationary points of the targeted pair are also clearly identified.


Assuntos
Citosina/química , DNA/química , Guanina/química , Ligação de Hidrogênio , Modelos Moleculares , Conformação de Ácido Nucleico
3.
Viruses ; 13(7)2021 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199077

RESUMO

Many viruses, especially RNA viruses, utilize programmed ribosomal frameshifting and/or stop codon readthrough in their expression, and in the decoding of a few a UGA is dynamically redefined to specify selenocysteine. This recoding can effectively increase viral coding capacity and generate a set ratio of products with the same N-terminal domain(s) but different C-terminal domains. Recoding can also be regulatory or generate a product with the non-universal 21st directly encoded amino acid. Selection for translation speed in the expression of many viruses at the expense of fidelity creates host immune defensive opportunities. In contrast to host opportunism, certain viruses, including some persistent viruses, utilize recoding or adventitious frameshifting as part of their strategy to evade an immune response or specific drugs. Several instances of recoding in small intensively studied viruses escaped detection for many years and their identification resolved dilemmas. The fundamental importance of ribosome ratcheting is consistent with the initial strong view of invariant triplet decoding which however did not foresee the possibility of transitory anticodon:codon dissociation. Deep level dynamics and structural understanding of recoding is underway, and a high level structure relevant to the frameshifting required for expression of the SARS CoV-2 genome has just been determined.


Assuntos
Vírus de DNA/genética , Vírus de DNA/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Evasão da Resposta Imune , Vírus de RNA/genética , Antivirais/farmacologia , Códon de Terminação , Vírus de DNA/efeitos dos fármacos , Mudança da Fase de Leitura do Gene Ribossômico , Antígenos de Histocompatibilidade Classe I/genética , Conformação de Ácido Nucleico , Peptídeos/imunologia , Biossíntese de Proteínas , Vírus de RNA/efeitos dos fármacos , Vírus de RNA/imunologia
4.
Molecules ; 26(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34204901

RESUMO

The topological properties of DNA molecules, supercoiling, knotting, and catenation, are intimately connected with essential biological processes, such as gene expression, replication, recombination, and chromosome segregation. Non-trivial DNA topologies present challenges to the molecular machines that process and maintain genomic information, for example, by creating unwanted DNA entanglements. At the same time, topological distortion can facilitate DNA-sequence recognition through localized duplex unwinding and longer-range loop-mediated interactions between the DNA sequences. Topoisomerases are a special class of essential enzymes that homeostatically manage DNA topology through the passage of DNA strands. The activities of these enzymes are generally investigated using circular DNA as a model system, in which case it is possible to directly assay the formation and relaxation of DNA supercoils and the formation/resolution of knots and catenanes. Some topoisomerases use ATP as an energy cofactor, whereas others act in an ATP-independent manner. The free energy of ATP hydrolysis can be used to drive negative and positive supercoiling or to specifically relax DNA topologies to levels below those that are expected at thermodynamic equilibrium. The latter activity, which is known as topology simplification, is thus far exclusively associated with type-II topoisomerases and it can be understood through insight into the detailed non-equilibrium behavior of type-II enzymes. We use a non-equilibrium topological-network approach, which stands in contrast to the equilibrium models that are conventionally used in the DNA-topology field, to gain insights into the rates that govern individual transitions between topological states. We anticipate that our quantitative approach will stimulate experimental work and the theoretical/computational modeling of topoisomerases and similar enzyme systems.


Assuntos
DNA Topoisomerases/metabolismo , DNA/química , DNA/metabolismo , Trifosfato de Adenosina/metabolismo , DNA Topoisomerases/química , Hidrólise , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica
5.
Nan Fang Yi Ke Da Xue Xue Bao ; 41(6): 960-964, 2021 Jun 20.
Artigo em Chinês | MEDLINE | ID: mdl-34238752

RESUMO

The development of DNA nanotechnology make it possible to artificially generate complex nucleic acid nanostructures with controllable sizes and shapes. DNA origami emerges as an effective and versatile approach to construct two- and three-dimensional programmable nanostructures, and represents a milestone in the development of structural DNA nanotechnology. Due to its high degree of controllable geometry, spatial addressability, easy chemical modification and good biocompatibility, DNA origami has great potentials for applications in many fields. In this review, we briefly summarize the applications of DNA origami in antigen-antibody interaction, targeted drug delivery and the synthesis of biomaterials.


Assuntos
DNA , Nanoestruturas , Sistemas de Liberação de Medicamentos , Nanotecnologia , Conformação de Ácido Nucleico
6.
Nat Commun ; 12(1): 4224, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244502

RESUMO

Nucleic acid-based constitutional dynamic networks (CDNs) have recently emerged as versatile tools to control a variety of catalytic processes. A key challenge in the application of these systems is achieving intercommunication between different CDNs to mimic the complex interlinked networks found in cellular biology. In particular, the possibility to interface photochemical 'energy-harvesting' processes with dark-operating 'metabolic' processes, in a similar way to plants, represents an up to now unexplored yet enticing research direction. The present study introduces two CDNs that allow the intercommunication of photocatalytic and dark-operating catalytic functions mediated by environmental components that facilitate the dynamic coupling of the networks. The dynamic feedback-driven intercommunication of the networks is accomplished via information transfer between the two CDNs effected by hairpin fuel strands in the environment of the system, leading to the coupling of the photochemical and dark-operating modules.


Assuntos
Materiais Biomiméticos/química , DNA Catalítico/química , Técnicas Genéticas , Processos Fotoquímicos , DNA Catalítico/genética , Luz , Conformação de Ácido Nucleico
7.
Nat Commun ; 12(1): 3397, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099665

RESUMO

It is known that an RNA's structure determines its biological function, yet current RNA structure probing methods only capture partial structure information. The ability to measure intact (i.e., full length) RNA structures will facilitate investigations of the functions and regulation mechanisms of small RNAs and identify short fragments of functional sites. Here, we present icSHAPE-MaP, an approach combining in vivo selective 2'-hydroxyl acylation and mutational profiling to probe intact RNA structures. We further showcase the RNA structural landscape of substrates bound by human Dicer based on the combination of RNA immunoprecipitation pull-down and icSHAPE-MaP small RNA structural profiling. We discover distinct structural categories of Dicer substrates in correlation to both their binding affinity and cleavage efficiency. And by tertiary structural modeling constrained by icSHAPE-MaP RNA structural data, we find the spatial distance measuring as an influential parameter for Dicer cleavage-site selection.


Assuntos
RNA Helicases DEAD-box/metabolismo , Conformação de Ácido Nucleico , RNA/química , Ribonuclease III/metabolismo , Biologia Computacional , RNA Helicases DEAD-box/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , RNA/genética , RNA/metabolismo , Sondas RNA , RNA-Seq , Ribonuclease III/genética , Especificidade por Substrato/genética
8.
Nat Commun ; 12(1): 3791, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145284

RESUMO

Particle fusion for single molecule localization microscopy improves signal-to-noise ratio and overcomes underlabeling, but ignores structural heterogeneity or conformational variability. We present a-priori knowledge-free unsupervised classification of structurally different particles employing the Bhattacharya cost function as dissimilarity metric. We achieve 96% classification accuracy on mixtures of up to four different DNA-origami structures, detect rare classes of origami occuring at 2% rate, and capture variation in ellipticity of nuclear pore complexes.


Assuntos
DNA/química , Poro Nuclear/química , Conformação de Ácido Nucleico , Imagem Individual de Molécula/métodos , Nanoestruturas/química , Razão Sinal-Ruído
9.
Nat Commun ; 12(1): 3557, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34117248

RESUMO

Bottom-up synthetic biology aims to engineer artificial cells capable of responsive behaviors by using a minimal set of molecular components. An important challenge toward this goal is the development of programmable biomaterials that can provide active spatial organization in cell-sized compartments. Here, we demonstrate the dynamic self-assembly of nucleic acid (NA) nanotubes inside water-in-oil droplets. We develop methods to encapsulate and assemble different types of DNA nanotubes from programmable DNA monomers, and demonstrate temporal control of assembly via designed pathways of RNA production and degradation. We examine the dynamic response of encapsulated nanotube assembly and disassembly with the support of statistical analysis of droplet images. Our study provides a toolkit of methods and components to build increasingly complex and functional NA materials to mimic life-like functions in synthetic cells.


Assuntos
DNA/química , Nanotecnologia/métodos , Nanotubos/química , Células Artificiais , Materiais Biocompatíveis , Emulsões , Gotículas Lipídicas , Substâncias Macromoleculares , Modelos Moleculares , Conformação de Ácido Nucleico , Oligonucleotídeos , Biologia Sintética , Água
10.
Nat Commun ; 12(1): 3368, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099723

RESUMO

Folding of RNA can produce elaborate tertiary structures, corresponding to their diverse roles in the regulation of biological activities. Direct observation of RNA structures at high resolution in their native form however remains a challenge. The large vestibule and the narrow constriction of a Mycobacterium smegmatis porin A (MspA) suggests a sensing mode called nanopore trapping/translocation, which clearly distinguishes between microRNA, small interfering RNA (siRNA), transfer RNA (tRNA) and 5 S ribosomal RNA (rRNA). To further profit from the acquired event characteristics, a custom machine learning algorithm is developed. Events from measurements with a mixture of RNA analytes can be automatically classified, reporting a general accuracy of ~93.4%. tRNAs, which possess a unique tertiary structure, report a highly distinguishable sensing feature, different from all other RNA types tested in this study. With this strategy, tRNAs from different sources are measured and a high structural conservation across different species is observed in single molecule.


Assuntos
Mycobacterium smegmatis/metabolismo , Nanoporos , Porinas/metabolismo , RNA/metabolismo , Aprendizado de Máquina , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , Simulação de Dinâmica Molecular , Peso Molecular , Mycobacterium smegmatis/genética , Conformação de Ácido Nucleico , Porinas/química , Porinas/genética , RNA/química , RNA/genética , Dobramento de RNA , Transporte de RNA , RNA Ribossômico 5S/química , RNA Ribossômico 5S/genética , RNA Ribossômico 5S/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo
11.
Proc Natl Acad Sci U S A ; 118(26)2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34185680

RESUMO

Translation of open reading frame 1b (ORF1b) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires a programmed -1 ribosomal frameshift (-1 PRF) promoted by an RNA pseudoknot. The extent to which SARS-CoV-2 replication may be sensitive to changes in -1 PRF efficiency is currently unknown. Through an unbiased, reporter-based high-throughput compound screen, we identified merafloxacin, a fluoroquinolone antibacterial, as a -1 PRF inhibitor for SARS-CoV-2. Frameshift inhibition by merafloxacin is robust to mutations within the pseudoknot region and is similarly effective on -1 PRF of other betacoronaviruses. Consistent with the essential role of -1 PRF in viral gene expression, merafloxacin impedes SARS-CoV-2 replication in Vero E6 cells, thereby providing proof-of-principle for targeting -1 PRF as a plausible and effective antiviral strategy for SARS-CoV-2 and other coronaviruses.


Assuntos
Antivirais/farmacologia , Mudança da Fase de Leitura do Gene Ribossômico/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Betacoronavirus , Chlorocebus aethiops , Fluoroquinolonas/farmacologia , Mudança da Fase de Leitura do Gene Ribossômico/genética , Mutação , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , SARS-CoV-2/fisiologia , Células Vero
12.
Proc Natl Acad Sci U S A ; 118(26)2021 06 29.
Artigo em Inglês | MEDLINE | ID: covidwho-1284758

RESUMO

Translation of open reading frame 1b (ORF1b) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires a programmed -1 ribosomal frameshift (-1 PRF) promoted by an RNA pseudoknot. The extent to which SARS-CoV-2 replication may be sensitive to changes in -1 PRF efficiency is currently unknown. Through an unbiased, reporter-based high-throughput compound screen, we identified merafloxacin, a fluoroquinolone antibacterial, as a -1 PRF inhibitor for SARS-CoV-2. Frameshift inhibition by merafloxacin is robust to mutations within the pseudoknot region and is similarly effective on -1 PRF of other betacoronaviruses. Consistent with the essential role of -1 PRF in viral gene expression, merafloxacin impedes SARS-CoV-2 replication in Vero E6 cells, thereby providing proof-of-principle for targeting -1 PRF as a plausible and effective antiviral strategy for SARS-CoV-2 and other coronaviruses.


Assuntos
Antivirais/farmacologia , Mudança da Fase de Leitura do Gene Ribossômico/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Betacoronavirus , Chlorocebus aethiops , Fluoroquinolonas/farmacologia , Mudança da Fase de Leitura do Gene Ribossômico/genética , Mutação , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , SARS-CoV-2/fisiologia , Células Vero
13.
PLoS Pathog ; 17(6): e1009596, 2021 06.
Artigo em Inglês | MEDLINE | ID: covidwho-1249581

RESUMO

The rapid evolution of RNA viruses has been long considered to result from a combination of high copying error frequencies during RNA replication, short generation times and the consequent extensive fixation of neutral or adaptive changes over short periods. While both the identities and sites of mutations are typically modelled as being random, recent investigations of sequence diversity of SARS coronavirus 2 (SARS-CoV-2) have identified a preponderance of C->U transitions, proposed to be driven by an APOBEC-like RNA editing process. The current study investigated whether this phenomenon could be observed in datasets of other RNA viruses. Using a 5% divergence filter to infer directionality, 18 from 36 datasets of aligned coding region sequences from a diverse range of mammalian RNA viruses (including Picornaviridae, Flaviviridae, Matonaviridae, Caliciviridae and Coronaviridae) showed a >2-fold base composition normalised excess of C->U transitions compared to U->C (range 2.1x-7.5x), with a consistently observed favoured 5' U upstream context. The presence of genome scale RNA secondary structure (GORS) was the only other genomic or structural parameter significantly associated with C->U/U->C transition asymmetries by multivariable analysis (ANOVA), potentially reflecting RNA structure dependence of sites targeted for C->U mutations. Using the association index metric, C->U changes were specifically over-represented at phylogenetically uninformative sites, potentially paralleling extensive homoplasy of this transition reported in SARS-CoV-2. Although mechanisms remain to be functionally characterised, excess C->U substitutions accounted for 11-14% of standing sequence variability of structured viruses and may therefore represent a potent driver of their sequence diversification and longer-term evolution.


Assuntos
Mamíferos/virologia , Mutação , Vírus de RNA/genética , SARS-CoV-2/genética , Desaminases APOBEC/metabolismo , Animais , Sequência de Bases , COVID-19/virologia , Citidina/genética , Dano ao DNA/fisiologia , Evolução Molecular , Regulação Viral da Expressão Gênica , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Humanos , Conformação de Ácido Nucleico , Filogenia , Edição de RNA/fisiologia , Vírus de RNA/classificação , RNA Viral/química , RNA Viral/genética , SARS-CoV-2/química , SARS-CoV-2/classificação , Análise de Sequência de RNA , Transcrição Genética/genética , Uridina/genética
14.
Nucleic Acids Res ; 49(11): 6128-6143, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34086938

RESUMO

Many non-coding RNAs with known functions are structurally conserved: their intramolecular secondary and tertiary interactions are maintained across evolutionary time. Consequently, the presence of conserved structure in multiple sequence alignments can be used to identify candidate functional non-coding RNAs. Here, we present a bioinformatics method that couples iterative homology search with covariation analysis to assess whether a genomic region has evidence of conserved RNA structure. We used this method to examine all unannotated regions of five well-studied fungal genomes (Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, Aspergillus fumigatus, and Schizosaccharomyces pombe). We identified 17 novel structurally conserved non-coding RNA candidates, which include four H/ACA box small nucleolar RNAs, four intergenic RNAs and nine RNA structures located within the introns and untranslated regions (UTRs) of mRNAs. For the two structures in the 3' UTRs of the metabolic genes GLY1 and MET13, we performed experiments that provide evidence against them being eukaryotic riboswitches.


Assuntos
RNA Fúngico/química , RNA não Traduzido/química , Regiões 3' não Traduzidas , Biologia Computacional/métodos , Genoma Fúngico , Íntrons , Lisina-tRNA Ligase/genética , Cadeias de Markov , Conformação de Ácido Nucleico , RNA Nucleolar Pequeno/química , Proteínas Ribossômicas/genética , Riboswitch , Alinhamento de Sequência , Tiorredoxinas/genética
15.
Nucleic Acids Res ; 49(11): 6375-6388, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34096583

RESUMO

Self-cleaving ribozymes are catalytic RNAs that cut themselves at a specific inter-nucleotide linkage. They serve as a model of RNA catalysis, and as an important tool in biotechnology. For most of the nine known structural classes of self-cleaving ribozymes, at least hundreds of examples are known, and some are present in multiple domains of life. By contrast, only four unique examples of the hairpin ribozyme class are known, despite its discovery in 1986. We bioinformatically predicted 941 unique hairpin ribozymes of a different permuted form from the four previously known hairpin ribozymes, and experimentally confirmed several diverse predictions. These results profoundly expand the number of natural hairpin ribozymes, enabling biochemical analysis based on natural sequences, and suggest that a distinct permuted form is more biologically relevant. Moreover, all novel hairpins were discovered in metatranscriptomes. They apparently reside in RNA molecules that vary both in size-from 381 to 5170 nucleotides-and in protein content. The RNA molecules likely replicate as circular single-stranded RNAs, and potentially provide a dramatic increase in diversity of such RNAs. Moreover, these organisms have eluded previous attempts to isolate RNA viruses from metatranscriptomes-suggesting a significant untapped universe of viruses or other organisms hidden within metatranscriptome sequences.


Assuntos
RNA Catalítico/química , RNA Circular/química , Biologia Computacional , Conformação de Ácido Nucleico , RNA Catalítico/metabolismo
16.
Nanoscale ; 13(25): 11197-11203, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34142687

RESUMO

We report experimental results on damage induced by ionizing radiation to DNA origami triangles which are commonly used prototypes for scaffolded DNA origami nanostructures. We demonstrate extreme stability of DNA origami upon irradiation, which is caused by (i) the multi-row design holding the shape of the origami even after severe damage to the scaffold DNA and (ii) the reduction of damage to the scaffold DNA due to the protective effect of the folded structure. With respect to damage induced by ionizing radiation, the protective effect of the structure is superior to that of a naturally paired DNA double helix. Present results allow estimating the stability of scaffolded DNA origami nanostructures in applications such as nanotechnology, pharmacy or in singulo molecular studies where they are exposed to ionizing radiation from natural and artificial sources. Additionally, possibilities are opened for scaffolded DNA use in the design of radiation-resistant and radio-sensitive materials.


Assuntos
Nanoestruturas , DNA , Nanotecnologia , Conformação de Ácido Nucleico , Radiação Ionizante
17.
Sci Signal ; 14(689)2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34131072

RESUMO

Capping of viral messenger RNAs is essential for efficient translation, for virus replication, and for preventing detection by the host cell innate response system. The SARS-CoV-2 genome encodes the 2'-O-methyltransferase nsp16, which, when bound to the coactivator nsp10, uses S-adenosylmethionine (SAM) as a donor to transfer a methyl group to the first ribonucleotide of the mRNA in the final step of viral mRNA capping. Here, we provide biochemical and structural evidence that this reaction requires divalent cations, preferably Mn2+, and a coronavirus-specific four-residue insert. We determined the x-ray structures of the SARS-CoV-2 2'-O-methyltransferase (the nsp16-nsp10 heterodimer) in complex with its reaction substrates, products, and divalent metal cations. These structural snapshots revealed that metal ions and the insert stabilize interactions between the capped RNA and nsp16, resulting in the precise alignment of the ribonucleotides in the active site. Comparison of available structures of 2'-O-methyltransferases with capped RNAs from different organisms revealed that the four-residue insert unique to coronavirus nsp16 alters the backbone conformation of the capped RNA in the binding groove, thereby promoting catalysis. This insert is highly conserved across coronaviruses, and its absence in mammalian methyltransferases makes this region a promising site for structure-guided drug design of selective coronavirus inhibitors.


Assuntos
COVID-19/virologia , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Humanos , Manganês/metabolismo , Metilação , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Capuzes de RNA/química , Capuzes de RNA/genética , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/genética , Transdução de Sinais , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
18.
Nat Commun ; 12(1): 3850, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158503

RESUMO

Three stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the TΨC-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 Å resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon.


Assuntos
Códon sem Sentido/genética , Códon de Terminação/genética , Escherichia coli/genética , Biossíntese de Proteínas/genética , RNA de Transferência/genética , Ribossomos/genética , Sequência de Bases , Sítios de Ligação/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Supressão Genética
19.
Nat Commun ; 12(1): 3877, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162884

RESUMO

Methylation is a prevalent post-transcriptional modification encountered in coding and non-coding RNA. For RNA methylation, cells use methyltransferases and small organic substances as methyl-group donors, such as S-adenosylmethionine (SAM). SAM and other nucleotide-derived cofactors are viewed as evolutionary leftovers from an RNA world, in which riboswitches have regulated, and ribozymes have catalyzed essential metabolic reactions. Here, we disclose the thus far unrecognized direct link between a present-day riboswitch and its inherent reactivity for site-specific methylation. The key is O6-methyl pre-queuosine (m6preQ1), a potentially prebiotic nucleobase which is recognized by the native aptamer of a preQ1 class I riboswitch. Upon binding, the transfer of the ligand's methyl group to a specific cytidine occurs, installing 3-methylcytidine (m3C) in the RNA pocket under release of pre-queuosine (preQ1). Our finding suggests that nucleic acid-mediated methylation is an ancient mechanism that has offered an early path for RNA epigenetics prior to the evolution of protein methyltransferases. Furthermore, our findings may pave the way for the development of riboswitch-descending methylation tools based on rational design as a powerful alternative to in vitro selection approaches.


Assuntos
Conformação de Ácido Nucleico , Nucleosídeo Q/química , RNA/química , Riboswitch , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Cinética , Metilação , Estrutura Molecular , Nucleosídeo Q/metabolismo , RNA/genética , RNA/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo
20.
Nat Commun ; 12(1): 3883, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162887

RESUMO

The Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication. The S. cerevisiae ORC binds to specific DNA sequences throughout the cell cycle but becomes active only when it binds to the replication initiator Cdc6. It has been unclear at the molecular level how Cdc6 activates ORC, converting it to an active recruiter of the Mcm2-7 hexamer, the core of the replicative helicase. Here we report the cryo-EM structure at 3.3 Å resolution of the yeast ORC-Cdc6 bound to an 85-bp ARS1 origin DNA. The structure reveals that Cdc6 contributes to origin DNA recognition via its winged helix domain (WHD) and its initiator-specific motif. Cdc6 binding rearranges a short α-helix in the Orc1 AAA+ domain and the Orc2 WHD, leading to the activation of the Cdc6 ATPase and the formation of the three sites for the recruitment of Mcm2-7, none of which are present in ORC alone. The results illuminate the molecular mechanism of a critical biochemical step in the licensing of eukaryotic replication origins.


Assuntos
Proteínas de Ciclo Celular/genética , Replicação do DNA/genética , DNA Fúngico/genética , Complexo de Reconhecimento de Origem/genética , Origem de Replicação/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , DNA Fúngico/química , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/metabolismo , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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