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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.
J Phys Chem B ; 125(28): 7613-7627, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34236202

RESUMO

Measuring the strength of the hydrogen bonds between DNA base pairs is of vital importance for understanding how our genetic code is physically accessed and recognized in cells, particularly during replication and transcription. Therefore, it is important to develop probes for these key hydrogen bonds (H-bonds) that dictate events critical to cellular function, such as the localized melting of DNA. The vibrations of carbonyl bonds are well-known probes of their H-bonding environment, and their signals can be observed with infrared (IR) spectroscopy. Yet, pinpointing a single bond of interest in the complex IR spectrum of DNA is challenging due to the large number of carbonyl signals that overlap with each other. Here, we develop a method using isotope editing and infrared (IR) spectroscopy to isolate IR signals from the thymine (T) C2═O carbonyl. We use solvatochromatic studies to show that the TC2═O signal's position in the IR spectrum is sensitive to the H-bonding capacity of the solvent. Our results indicate that C2═O of a single T base within DNA duplexes experiences weak H-bonding interactions. This finding is consistent with the existence of a third, noncanonical CH···O H-bond between adenine and thymine in both Watson-Crick and Hoogsteen base pairs in DNA.


Assuntos
DNA , Isótopos , Hidrogênio , Ligação de Hidrogênio , Análise Espectral
3.
Nucleic Acids Res ; 49(8): 4281-4293, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33856457

RESUMO

Deazapurine nucleosides such as 3-deazaadenosine (c3A) are crucial for atomic mutagenesis studies of functional RNAs. They were the key for our current mechanistic understanding of ribosomal peptide bond formation and of phosphodiester cleavage in recently discovered small ribozymes, such as twister and pistol RNAs. Here, we present a comprehensive study on the impact of c3A and the thus far underinvestigated 3-deazaguanosine (c3G) on RNA properties. We found that these nucleosides can decrease thermodynamic stability of base pairing to a significant extent. The effects are much more pronounced for 3-deazapurine nucleosides compared to their constitutional isomers of 7-deazapurine nucleosides (c7G, c7A). We furthermore investigated base pair opening dynamics by solution NMR spectroscopy and revealed significantly enhanced imino proton exchange rates. Additionally, we solved the X-ray structure of a c3A-modified RNA and visualized the hydration pattern of the minor groove. Importantly, the characteristic water molecule that is hydrogen-bonded to the purine N3 atom and always observed in a natural double helix is lacking in the 3-deazapurine-modified counterpart. Both, the findings by NMR and X-ray crystallographic methods hence provide a rationale for the reduced pairing strength. Taken together, our comparative study is a first major step towards a comprehensive understanding of this important class of nucleoside modifications.


Assuntos
Estabilidade de RNA , RNA/química , Tubercidina/química , Pareamento de Bases , Cristalografia por Raios X , Mutagênese , Purinas/química , RNA/genética , Termodinâmica
4.
Nat Commun ; 11(1): 5750, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33188169

RESUMO

Nuclear export complexes composed of rev response element (RRE) ribonucleic acid (RNA) and multiple molecules of rev protein are promising targets for the development of therapeutic strategies against human immunodeficiency virus type 1 (HIV-1), but their assembly remains poorly understood. Using native mass spectrometry, we show here that rev initially binds to the upper stem of RRE IIB, from where it is relayed to binding sites that allow for rev dimerization. The newly discovered binding region implies initial rev recognition by nucleotides that are not part of the internal loop of RRE stem IIB RNA, which was previously identified as the preferred binding region. Our study highlights the unique capability of native mass spectrometry to separately study the binding interfaces of RNA/protein complexes of different stoichiometry, and provides a detailed understanding of the mechanism of RRE/rev association with implications for the rational design of potential drugs against HIV-1 infection.


Assuntos
HIV-1/metabolismo , RNA Viral/metabolismo , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Sítios de Ligação , Genes env , HIV-1/química , HIV-1/genética , Espectrometria de Massas , Conformação de Ácido Nucleico , Multimerização Proteica , RNA Viral/química , RNA Viral/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/química
5.
Nucleic Acids Res ; 48(21): 12365-12379, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33104789

RESUMO

2'-O-Methyl (Nm) is a highly abundant post-transcriptional RNA modification that plays important biological roles through mechanisms that are not entirely understood. There is evidence that Nm can alter the biological activities of RNAs by biasing the ribose sugar pucker equilibrium toward the C3'-endo conformation formed in canonical duplexes. However, little is known about how Nm might more broadly alter the dynamic ensembles of flexible RNAs containing bulges and internal loops. Here, using NMR and the HIV-1 transactivation response (TAR) element as a model system, we show that Nm preferentially stabilizes alternative secondary structures in which the Nm-modified nucleotides are paired, increasing both the abundance and lifetime of low-populated short-lived excited states by up to 10-fold. The extent of stabilization increased with number of Nm modifications and was also dependent on Mg2+. Through phi-value analysis, the Nm modification also provided rare insights into the structure of the transition state for conformational exchange. Our results suggest that Nm could alter the biological activities of Nm-modified RNAs by modulating their secondary structural ensembles as well as establish the utility of Nm as a tool for the discovery and characterization of RNA excited state conformations.


Assuntos
Repetição Terminal Longa de HIV , Magnésio/química , Processamento Pós-Transcricional do RNA , RNA Viral/química , Pareamento de Bases , Cátions Bivalentes , Teoria da Densidade Funcional , HIV-1/química , Magnésio/metabolismo , Espectroscopia de Ressonância Magnética , Metilação , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Viral/genética , RNA Viral/metabolismo , Termodinâmica
6.
Angew Chem Int Ed Engl ; 59(39): 17062-17069, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32558232

RESUMO

We present the access to [5-19 F, 5-13 C]-uridine and -cytidine phosphoramidites for the production of site-specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5-19 F, 5-13 C]-pyrimidine labels into five RNAs-the 30 nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61 nt human hepatitis B virus ϵ (hHBV ϵ) RNA, the 49 nt SAM VI riboswitch aptamer domain from B. angulatum, the 29 nt apical stem loop of the pre-microRNA (miRNA) 21 and the 59 nt full length pre-miRNA 21. The main stimulus to introduce the aromatic 19 F-13 C-spin topology into RNA comes from a work of Boeszoermenyi et al., in which the dipole-dipole interaction and the chemical shift anisotropy relaxation mechanisms cancel each other leading to advantageous TROSY properties shown for aromatic protein sidechains. This aromatic 13 C-19 F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5-19 F, 5-13 C]/[5-19 F] pyrimidine labeling. For the 61 nt hHBV ϵ we find a beneficial 19 F-13 C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [19 F, 13 C]-labeling of the SAM VI aptamer domain and the pre-miRNA 21 further opens the possibility to use the biorthogonal stable isotope reporter nuclei in in vivo NMR to observe ligand binding and microRNA processing in a biological relevant setting.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Pirimidinas/química , RNA Viral/química , Isótopos de Carbono , Flúor
7.
Sci Rep ; 10(1): 5653, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221355

RESUMO

Morphine and structurally-derived compounds are µ opioid receptor (µOR) agonists, and the most effective analgesic drugs. However, their usefulness is limited by serious side effects, including dependence and abuse potential. The N-substituent in morphinans plays an important role in opioid activities in vitro and in vivo. This study presents the synthesis and pharmacological evaluation of new N-phenethyl substituted 14-O-methylmorphinan-6-ones. Whereas substitution of the N-methyl substituent in morphine (1) and oxymorphone (2) by an N-phenethyl group enhances binding affinity, selectivity and agonist potency at the µOR of 1a and 2a, the N-phenethyl substitution in 14-methoxy-N-methylmorphinan-6-ones (3 and 4) converts selective µOR ligands into dual µ/δOR agonists (3a and 4a). Contrary to N-methylmorphinans 1-4, the N-phenethyl substituted morphinans 1a-4a produce effective and potent antinociception without motor impairment in mice. Using docking and molecular dynamics simulations with the µOR, we establish that N-methylmorphinans 1-4 and their N-phenethyl counterparts 1a-4a share several essential receptor-ligand interactions, but also interaction pattern differences related to specific structural features, thus providing a structural basis for their pharmacological profiles. The emerged structure-activity relationships in this class of morphinans provide important information for tuning in vitro and in vivo opioid activities towards discovery of effective and safer analgesics.


Assuntos
Analgésicos Opioides/farmacologia , Morfinanos/farmacologia , Receptores Opioides delta/agonistas , Receptores Opioides delta/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetulus , Humanos , Ligantes , Masculino , Camundongos , Morfina/farmacologia , Relação Estrutura-Atividade
8.
Nucleic Acids Res ; 48(2): 949-961, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31754719

RESUMO

RNA aptamers-artificially created RNAs with high affinity and selectivity for their target ligand generated from random sequence pools-are versatile tools in the fields of biotechnology and medicine. On a more fundamental level, they also further our general understanding of RNA-ligand interactions e. g. in regard to the relationship between structural complexity and ligand affinity and specificity, RNA structure and RNA folding. Detailed structural knowledge on a wide range of aptamer-ligand complexes is required to further our understanding of RNA-ligand interactions. Here, we present the atomic resolution structure of an RNA-aptamer binding to the fluorescent xanthene dye tetramethylrhodamine. The high resolution structure, solved by NMR-spectroscopy in solution, reveals binding features both common and different from the binding mode of other aptamers with affinity for ligands carrying planar aromatic ring systems such as the malachite green aptamer which binds to the tetramethylrhodamine related dye malachite green or the flavin mononucleotide aptamer.


Assuntos
Aptâmeros de Nucleotídeos/química , Conformação de Ácido Nucleico , RNA/química , Rodaminas/química , Ligantes , Espectroscopia de Ressonância Magnética , Dobramento de RNA
9.
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
10.
J Am Chem Soc ; 141(51): 19988-19993, 2019 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-31826614

RESUMO

N6-Methyladenosine (m6A) is an abundant epitranscriptomic modification that plays important roles in many aspects of RNA metabolism. While m6A is thought to mainly function by recruiting reader proteins to specific RNA sites, the modification can also reshape RNA-protein and RNA-RNA interactions by altering RNA structure mainly by destabilizing base pairing. Little is known about how m6A and other epitranscriptomic modifications might affect the kinetic rates of RNA folding and other conformational transitions that are also important for cellular activity. Here, we used NMR R1ρ relaxation dispersion and chemical exchange saturation transfer to noninvasively and site-specifically measure nucleic acid hybridization kinetics. The methodology was validated on two DNA duplexes and then applied to examine how a single m6A alters the hybridization kinetics in two RNA duplexes. The results show that m6A minimally impacts the rate constant for duplex dissociation, changing koff by ∼1-fold but significantly slows the rate of duplex annealing, decreasing kon by ∼7-fold. A reduction in the annealing rate was observed robustly for two different sequence contexts at different temperatures, both in the presence and absence of Mg2+. We propose that rotation of the N6-methyl group from the preferred syn conformation in the unpaired nucleotide to the energetically disfavored anti conformation required for Watson-Crick pairing is responsible for the reduced annealing rate. The results help explain why in mRNA m6A slows down tRNA selection and more generally suggest that m6A may exert cellular functions by reshaping the kinetics of RNA conformational transitions.


Assuntos
Adenosina/análogos & derivados , Ressonância Magnética Nuclear Biomolecular , RNA/química , Adenosina/análise , Adenosina/metabolismo , RNA/metabolismo
11.
Nucleic Acids Res ; 47(21): 11430-11440, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31665419

RESUMO

Although group II intron ribozymes are intensively studied the question how structural dynamics affects splicing catalysis has remained elusive. We report for the first time that the group II intron domain 6 exists in a secondary structure equilibrium between a single- and a two-nucleotide bulge conformation, which is directly linked to a switch between sugar puckers of the branch site adenosine. Our study determined a functional sugar pucker equilibrium between the transesterification active C2'-endo conformation of the branch site adenosine in the 1nt bulge and an inactive C3'-endo state in the 2nt bulge fold, allowing the group II intron to switch its activity from the branching to the exon ligation step. Our detailed NMR spectroscopic investigation identified magnesium (II) ions and the branching reaction as regulators of the equilibrium populations. The tuneable secondary structure/sugar pucker equilibrium supports a conformational selection mechanism to up- and downregulate catalytically active and inactive states of the branch site adenosine to orchestrate the multi-step splicing process. The conformational dynamics of group II intron domain 6 is also proposed to be a key aspect for the directionality selection in reversible splicing.


Assuntos
Íntrons/genética , Conformação de Ácido Nucleico , Splicing de RNA/fisiologia , RNA/química , Açúcares/química , Sítios de Ligação , Carboidratos/química , Magnésio/química , Espectroscopia de Ressonância Magnética , RNA/metabolismo , Açúcares/metabolismo
12.
J Magn Reson ; 308: 106589, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31539864

RESUMO

NMR relaxation dispersion studies have shown that Watson-Crick G-C and A-T base pairs in duplex DNA exist in dynamic equilibrium with their Hoogsteen counterparts. Hoogsteen base pairs form through concurrent rotation of the purine base about the glycosidic bond from an anti to a syn conformation and constriction of the C1'-C1' distance across the base pair by ∼2 Što allow Hoogsteen type hydrogen bonding. Owing to their unique structure, Hoogsteen base pairs can play important roles in DNA recognition, the accommodation, recognition, and repair of DNA damage, and in DNA replication. NMR relaxation dispersion experiments targeting imino nitrogen and protonated base and sugar carbons have provided insights into many structural features of transient Hoogsteen base pairs, including one of two predicted hydrogen bonds involving (G)N7···H-N3(C)+ and (A)N7···H-N3(T). Here, through measurement of cytosine amino (N4) R1ρ relaxation dispersion, we provide direct evidence for the second (G)O6···H2-N4(C)+ hydrogen bond in G(syn)-C+ transient Hoogsteen base pairs. The utility of cytosine N4 R1ρ relaxation dispersion as a new sensitive probe of transient Hoogsteen base pairs, and cytosine dynamics in general, is further demonstrated by measuring G(syn)-C+ Hoogsteen exchange near neutral pH and in the context of the naturally occurring DNA modification 5-methyl cytosine (m5C), in DNA samples prepared using chemical synthesis and a 15N labeled m5C phosphoramidite.


Assuntos
Pareamento de Bases , Citosina/química , DNA/química , Ligação de Hidrogênio , Nitrogênio/química , Adenosina/química , Teoria da Densidade Funcional , Epigênese Genética , Guanina/química , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética/métodos , Oligonucleotídeos/química , Timina/química
13.
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
14.
Angew Chem Int Ed Engl ; 58(31): 10756-10760, 2019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31115943

RESUMO

The B12 cofactors instill a natural curiosity regarding the primordial selection and evolution of their corrin ligand. Surprisingly, this important natural macrocycle has evaded molecular scrutiny, and its specific role in predisposing the incarcerated cobalt ion for organometallic catalysis has remained obscure. Herein, we report the biosynthesis of the cobalt-free B12 corrin moiety, hydrogenobyric acid (Hby), a compound crafted through pathway redesign. Detailed insights from single-crystal X-ray and solution structures of Hby have revealed a distorted helical cavity, redefining the pattern for binding cobalt ions. Consequently, the corrin ligand coordinates cobalt ions in desymmetrized "entatic" states, thereby promoting the activation of B12 -cofactors for their challenging chemical transitions. The availability of Hby also provides a route to the synthesis of transition metal analogues of B12 .


Assuntos
Corrinoides/biossíntese , Uroporfirinas/metabolismo , Vitamina B 12/metabolismo , Biocatálise , Cobalto/química , Cobalto/metabolismo , Corrinoides/química , Ligantes , Estrutura Molecular , Uroporfirinas/química , Vitamina B 12/química
15.
Org Lett ; 21(11): 3900-3903, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31081638

RESUMO

3-Deazaguanosine (c3G) and phosphoramidite derivatives thereof that allow incorporation of this modification into RNA are needed for atomic mutagenesis experiments to explore mechanistic aspects of ribozyme catalysis. Here, we report a practical synthesis for c3G phosphoramidites from inexpensive starting materials. The key reaction sequence is a silyl-Hilbert-Johnson nucleosidation followed by Hartwig-Buchwald cross-coupling to achieve the N2-phenoxyacetyl protected c3G nucleoside.


Assuntos
Guanosina/análogos & derivados , Compostos Organofosforados/síntese química , RNA/química , Técnicas de Síntese em Fase Sólida , Guanosina/química , Estrutura Molecular , Compostos Organofosforados/química
16.
Biomol NMR Assign ; 13(2): 281-286, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31030336

RESUMO

Ligand binding RNAs such as artificially created RNA-aptamers are structurally highly diverse. Therefore, they represent important model systems for investigating RNA-folding, RNA-dynamics and the molecular recognition of chemically very different ligands, ranging from small molecules to whole cells. High-resolution structures of RNA-aptamers in complex with their cognate ligands often reveal unexpected tertiary structure elements. Recent studies on different classes of aptamers binding the nucleotide triphosphate GTP as a ligand showed that these systems not only differ widely in binding affinity but also in their ligand binding modes and structural complexity. We initiated the NMR-based structure determination of the high-affinity binding GTP-aptamer 9-12 in order to gain further insights into the diversity of ligand binding modes and structural variability of those aptamers. Here, we report 1H, 13C and 15N resonance assignments for the GTP 9-12-aptamer bound to GTP as the prerequisite for the structure determination by solution NMR.


Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Guanosina Trifosfato/metabolismo , Ressonância Magnética Nuclear Biomolecular , Aptâmeros de Nucleotídeos/genética , Sequência de Bases
17.
Genes (Basel) ; 10(2)2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30691071

RESUMO

RNA modifications are crucial factors for efficient protein synthesis. All classes of RNAs that are involved in translation are modified to different extents. Recently, mRNA modifications and their impact on gene regulation became a focus of interest because they can exert a variety of effects on the fate of mRNAs. mRNA modifications within coding sequences can either directly or indirectly interfere with protein synthesis. In order to investigate the roles of various natural occurring modified nucleotides, we site-specifically introduced them into the coding sequence of reporter mRNAs and subsequently translated them in HEK293T cells. The analysis of the respective protein products revealed a strong position-dependent impact of RNA modifications on translation efficiency and accuracy. Whereas a single 5-methylcytosine (m5C) or pseudouridine () did not reduce product yields, N¹-methyladenosine (m¹A) generally impeded the translation of the respective modified mRNA. An inhibitory effect of 2'O-methlyated nucleotides (Nm) and N6-methyladenosine (m6A) was strongly dependent on their position within the codon. Finally, we could not attribute any miscoding potential to the set of mRNA modifications tested in HEK293T cells.


Assuntos
Elongação Traducional da Cadeia Peptídica , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , 5-Metilcitosina/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Pseudouridina/metabolismo , RNA Mensageiro/metabolismo
18.
Nucleic Acids Res ; 47(5): 2654-2665, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30590743

RESUMO

S-adenosylmethionine (SAM) is a central metabolite since it is used as a methyl group donor in many different biochemical reactions. Many bacteria control intracellular SAM concentrations using riboswitch-based mechanisms. A number of structurally different riboswitch families specifically bind to SAM and mainly regulate the transcription or the translation of SAM-biosynthetic enzymes. In addition, a highly specific riboswitch class recognizes S-adenosylhomocysteine (SAH)-the product of SAM-dependent methyl group transfer reactions-and regulates enzymes responsible for SAH hydrolysis. High-resolution structures are available for many of these riboswitch classes and illustrate how they discriminate between the two structurally similar ligands SAM and SAH. The so-called SAM/SAH riboswitch class binds both ligands with similar affinities and is structurally not yet characterized. Here, we present a high-resolution nuclear magnetic resonance structure of a member of the SAM/SAH-riboswitch class in complex with SAH. Ligand binding induces pseudoknot formation and sequestration of the ribosome binding site. Thus, the SAM/SAH-riboswitches are translational 'OFF'-switches. Our results establish a structural basis for the unusual bispecificity of this riboswitch class. In conjunction with genomic data our structure suggests that the SAM/SAH-riboswitches might be an evolutionary late invention and not a remnant of a primordial RNA-world as suggested for other riboswitches.


Assuntos
Biossíntese de Proteínas , Riboswitch/genética , S-Adenosil-Homocisteína/química , S-Adenosilmetionina/química , Evolução Molecular , Genômica , Ligantes , RNA/química , RNA/genética , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo
19.
Nat Commun ; 9(1): 4865, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30451861

RESUMO

The precise interplay between the mRNA codon and the tRNA anticodon is crucial for ensuring efficient and accurate translation by the ribosome. The insertion of RNA nucleobase derivatives in the mRNA allowed us to modulate the stability of the codon-anticodon interaction in the decoding site of bacterial and eukaryotic ribosomes, allowing an in-depth analysis of codon recognition. We found the hydrogen bond between the N1 of purines and the N3 of pyrimidines to be sufficient for decoding of the first two codon nucleotides, whereas adequate stacking between the RNA bases is critical at the wobble position. Inosine, found in eukaryotic mRNAs, is an important example of destabilization of the codon-anticodon interaction. Whereas single inosines are efficiently translated, multiple inosines, e.g., in the serotonin receptor 5-HT2C mRNA, inhibit translation. Thus, our results indicate that despite the robustness of the decoding process, its tolerance toward the weakening of codon-anticodon interactions is limited.


Assuntos
2-Aminopurina/análogos & derivados , Anticódon/química , Códon/química , Inosina/metabolismo , Biossíntese de Proteínas , Receptor 5-HT2C de Serotonina/genética , 2-Aminopurina/química , 2-Aminopurina/metabolismo , Anticódon/metabolismo , Bacteriófago T7/genética , Bacteriófago T7/metabolismo , Sequência de Bases , Códon/metabolismo , Citidina/análogos & derivados , Citidina/genética , Citidina/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Ligação de Hidrogênio , Inosina/genética , Piridonas/química , Piridonas/metabolismo , RNA de Transferência de Glicina/genética , RNA de Transferência de Glicina/metabolismo , Receptor 5-HT2C de Serotonina/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
20.
Chemistry ; 24(71): 18903-18906, 2018 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-30300940

RESUMO

Watson-Crick like G-U mismatches with tautomeric Genol or Uenol bases can evade fidelity checkpoints and thereby contribute to translational errors. The 5-oxyacetic acid uridine (cmo5 U) modification is a base modification at the wobble position on tRNAs and is presumed to expand the decoding capability of tRNA at this position by forming Watson-Crick like cmo5 Uenol -G mismatches. A detailed investigation on the influence of the cmo5 U modification on structural and dynamic features of RNA was carried out by using solution NMR spectroscopy and UV melting curve analysis. The introduction of a stable isotope labeled variant of the cmo5 U modifier allowed the application of relaxation dispersion NMR to probe the potentially formed Watson-Crick like cmo5 Uenol -G base pair. Surprisingly, we find that at neutral pH, the modification promotes transient formation of anionic Watson-Crick like cmo5 U- -G, and not enolic base pairs. Our results suggest that recoding is mediated by an anionic Watson-Crick like species, as well as bring an interesting aspect of naturally occurring RNA modifications into focus-the fine tuning of nucleobase properties leading to modulation of the RNA structural landscape by adoption of alternative base pairing patterns.

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