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1.
Nat Commun ; 12(1): 5201, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34465779

RESUMO

N6-methyladenosine (m6A) is a post-transcriptional modification that controls gene expression by recruiting proteins to RNA sites. The modification also slows biochemical processes through mechanisms that are not understood. Using temperature-dependent (20°C-65°C) NMR relaxation dispersion, we show that m6A pairs with uridine with the methylamino group in the anti conformation to form a Watson-Crick base pair that transiently exchanges on the millisecond timescale with a singly hydrogen-bonded low-populated (1%) mismatch-like conformation in which the methylamino group is syn. This ability to rapidly interchange between Watson-Crick or mismatch-like forms, combined with different syn:anti isomer preferences when paired (~1:100) versus unpaired (~10:1), explains how m6A robustly slows duplex annealing without affecting melting at elevated temperatures via two pathways in which isomerization occurs before or after duplex annealing. Our model quantitatively predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions, and provides an explanation for why the modification robustly slows diverse cellular processes.


Assuntos
Adenosina/análogos & derivados , DNA/química , DNA/metabolismo , Adenosina/química , Adenosina/genética , Adenosina/metabolismo , Pareamento de Bases , DNA/genética , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Processamento Pós-Transcricional do RNA , Uridina/química , Uridina/genética , Uridina/metabolismo
2.
RNA ; 27(1): 12-26, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33028652

RESUMO

Identifying small molecules that selectively bind an RNA target while discriminating against all other cellular RNAs is an important challenge in RNA-targeted drug discovery. Much effort has been directed toward identifying drug-like small molecules that minimize electrostatic and stacking interactions that lead to nonspecific binding of aminoglycosides and intercalators to many stem-loop RNAs. Many such compounds have been reported to bind RNAs and inhibit their cellular activities. However, target engagement and cellular selectivity assays are not routinely performed, and it is often unclear whether functional activity directly results from specific binding to the target RNA. Here, we examined the propensities of three drug-like compounds, previously shown to bind and inhibit the cellular activities of distinct stem-loop RNAs, to bind and inhibit the cellular activities of two unrelated HIV-1 stem-loop RNAs: the transactivation response element (TAR) and the rev response element stem IIB (RREIIB). All compounds bound TAR and RREIIB in vitro, and two inhibited TAR-dependent transactivation and RRE-dependent viral export in cell-based assays while also exhibiting off-target interactions consistent with nonspecific activity. A survey of X-ray and NMR structures of RNA-small molecule complexes revealed that aminoglycosides and drug-like molecules form hydrogen bonds with functional groups commonly accessible in canonical stem-loop RNA motifs, in contrast to ligands that specifically bind riboswitches. Our results demonstrate that drug-like molecules can nonspecifically bind stem-loop RNAs most likely through hydrogen bonding and electrostatic interactions and reinforce the importance of assaying for off-target interactions and RNA selectivity in vitro and in cells when assessing novel RNA-binders.


Assuntos
Aminoglicosídeos/farmacologia , Genes env/efeitos dos fármacos , Repetição Terminal Longa de HIV/efeitos dos fármacos , RNA Viral/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Aminoglicosídeos/química , Aminoglicosídeos/metabolismo , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Bioensaio , Descoberta de Drogas , HIV-1/efeitos dos fármacos , HIV-1/genética , HIV-1/metabolismo , Humanos , Ligação de Hidrogênio , Isoquinolinas/química , Isoquinolinas/metabolismo , Isoquinolinas/farmacologia , Conformação de Ácido Nucleico , Pentamidina/química , Pentamidina/metabolismo , Pentamidina/farmacologia , RNA Viral/genética , RNA Viral/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Eletricidade Estática , Ativação Transcricional/efeitos dos fármacos , Ioimbina/química , Ioimbina/metabolismo , Ioimbina/farmacologia
3.
Cell Rep ; 30(8): 2472-2480.e4, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32101729

RESUMO

Low-abundance short-lived non-native conformations referred to as excited states (ESs) are increasingly observed in vitro and implicated in the folding and biological activities of regulatory RNAs. We developed an approach for assessing the relative abundance of RNA ESs within the functional cellular context. Nuclear magnetic resonance (NMR) spectroscopy was used to estimate the degree to which substitution mutations bias conformational equilibria toward the inactive ES in vitro. The cellular activity of the ES-stabilizing mutants was used as an indirect measure of the conformational equilibria within the functional cellular context. Compensatory mutations that restore the ground-state conformation were used to control for changes in sequence. Using this approach, we show that the ESs of two regulatory RNAs from HIV-1, the transactivation response element (TAR) and the Rev response element (RRE), likely form in cells with abundances comparable to those measured in vitro, and their targeted stabilization may provide an avenue for developing anti-HIV therapeutics.


Assuntos
Células/metabolismo , Conformação de Ácido Nucleico , Microambiente Celular , Genes env , Células HEK293 , Células HeLa , Humanos , Estabilidade de RNA
4.
PLoS One ; 14(12): e0224850, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31825959

RESUMO

N6-methyladenosine (m6A) is a ubiquitous RNA post-transcriptional modification found in coding as well as non-coding RNAs. m6A has also been found in viral RNAs where it is proposed to modulate host-pathogen interactions. Two m6A sites have been reported in the HIV-1 Rev response element (RRE) stem IIB, one of which was shown to enhance binding to the viral protein Rev and viral RNA export. However, because these m6A sites have not been observed in other studies mapping m6A in HIV-1 RNA, their significance remains to be firmly established. Here, using optical melting experiments, NMR spectroscopy, and in vitro binding assays, we show that m6A minimally impacts the stability, structure, and dynamics of RRE stem IIB as well as its binding affinity to the Rev arginine-rich-motif (ARM) in vitro. Our results indicate that if present in stem IIB, m6A is unlikely to substantially alter the conformational properties of the RNA. Our results add to a growing view that the impact of m6A on RNA depends on sequence context and Mg2+.


Assuntos
Adenosina/análogos & derivados , RNA Viral/química , RNA Viral/metabolismo , Elementos de Resposta , Produtos do Gene rev do Vírus da Imunodeficiência Humana/química , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Adenosina/química , Pareamento de Bases , Sequência de Bases , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Ligação Proteica , RNA Viral/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética
5.
Nucleic Acids Res ; 47(13): 7105-7117, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31199872

RESUMO

The HIV-1 Rev response element (RRE) RNA element mediates the nuclear export of intron containing viral RNAs by forming an oligomeric complex with the viral protein Rev. Stem IIB and nearby stem II three-way junction nucleate oligomerization through cooperative binding of two Rev molecules. Conformational flexibility at this RRE region has been shown to be important for Rev binding. However, the nature of the flexibility has remained elusive. Here, using NMR relaxation dispersion, including a new strategy for directly observing transient conformational states in large RNAs, we find that stem IIB alone or when part of the larger RREII three-way junction robustly exists in dynamic equilibrium with non-native excited state (ES) conformations that have a combined population of ∼20%. The ESs disrupt the Rev-binding site by changing local secondary structure, and their stabilization via point substitution mutations decreases the binding affinity to the Rev arginine-rich motif (ARM) by 15- to 80-fold. The ensemble clarifies the conformational flexibility observed in stem IIB, reveals long-range conformational coupling between stem IIB and the three-way junction that may play roles in cooperative Rev binding, and also identifies non-native RRE conformational states as new targets for the development of anti-HIV therapeutics.


Assuntos
Genes env , HIV-1/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Magnésio/metabolismo , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo
6.
Nucleic Acids Res ; 47(10): 5126-5140, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30916331

RESUMO

RecA is essential to recombinational DNA repair in which RecA filaments mediate the homologous DNA pairing and strand exchange. Both RecA filament assembly and the subsequent DNA strand exchange are directional. Here, we demonstrate that the polarity of DNA strand exchange is embedded within RecA filaments even in the absence of ATP hydrolysis, at least over short DNA segments. Using single-molecule tethered particle motion, we show that successful strand exchange in the presence of ATP proceeds with a 5'-to-3' polarity, as demonstrated previously. RecA filaments prepared with ATPγS also exhibit a 5'-to-3' progress of strand exchange, suggesting that the polarity is not determined by RecA disassembly and/or ATP hydrolysis. RecAΔC17 mutants, lacking a C-terminal autoregulatory flap, also promote strand exchange in a 5'-to-3' polarity in ATPγS, a polarity that is largely lost with this RecA variant when ATP is hydrolyzed. We propose that there is an inherent strand exchange polarity mediated by the structure of the RecA filament groove, associated by conformation changes propagated in a polar manner as DNA is progressively exchanged. ATP hydrolysis is coupled to polar strand exchange over longer distances, and its contribution to the polarity requires an intact RecA C-terminus.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Recombinases Rec A/metabolismo , Trifosfato de Adenosina/análogos & derivados , DNA Bacteriano/genética , DNA de Cadeia Simples , Escherichia coli/metabolismo , Hidrólise , Íons , Cinética , Magnésio/química , Nucleoproteínas/metabolismo , Domínios Proteicos
7.
Nature ; 554(7691): 195-201, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29420478

RESUMO

Tautomeric and anionic Watson-Crick-like mismatches have important roles in replication and translation errors through mechanisms that are not fully understood. Here, using NMR relaxation dispersion, we resolve a sequence-dependent kinetic network connecting G•T/U wobbles with three distinct Watson-Crick mismatches: two rapidly exchanging tautomeric species (Genol•T/UG•Tenol/Uenol; population less than 0.4%) and one anionic species (G•T-/U-; population around 0.001% at neutral pH). The sequence-dependent tautomerization or ionization step was inserted into a minimal kinetic mechanism for correct incorporation during replication after the initial binding of the nucleotide, leading to accurate predictions of the probability of dG•dT misincorporation across different polymerases and pH conditions and for a chemically modified nucleotide, and providing mechanisms for sequence-dependent misincorporation. Our results indicate that the energetic penalty for tautomerization and/or ionization accounts for an approximately 10-2 to 10-3-fold discrimination against misincorporation, which proceeds primarily via tautomeric dGenol•dT and dG•dTenol, with contributions from anionic dG•dT- dominant at pH 8.4 and above or for some mutagenic nucleotides.


Assuntos
Pareamento Incorreto de Bases , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , DNA/química , Guanina/metabolismo , Mutagênese , Timina/metabolismo , Animais , Ânions , Pareamento Incorreto de Bases/genética , DNA/genética , Guanina/química , Humanos , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Probabilidade , Ratos , Timina/química
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