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1.
Nat Commun ; 12(1): 4723, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354064

RESUMO

Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.


Assuntos
Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Ribossomos/química , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo
3.
Chembiochem ; 22(2): 423-433, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32794266

RESUMO

We report here the nuclear magnetic resonance 19 F screening of 14 RNA targets with different secondary and tertiary structure to systematically assess the druggability of RNAs. Our RNA targets include representative bacterial riboswitches that naturally bind with nanomolar affinity and high specificity to cellular metabolites of low molecular weight. Based on counter-screens against five DNAs and five proteins, we can show that RNA can be specifically targeted. To demonstrate the quality of the initial fragment library that has been designed for easy follow-up chemistry, we further show how to increase binding affinity from an initial fragment hit by chemistry that links the identified fragment to the intercalator acridine. Thus, we achieve low-micromolar binding affinity without losing binding specificity between two different terminator structures.

4.
Nucleic Acids Res ; 48(22): 12415-12435, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33167030

RESUMO

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.


Assuntos
COVID-19/prevenção & controle , Espectroscopia de Ressonância Magnética/métodos , Conformação de Ácido Nucleico , RNA Viral/química , SARS-CoV-2/genética , Regiões 3' não Traduzidas/genética , Sequência de Bases , COVID-19/epidemiologia , COVID-19/virologia , Mudança da Fase de Leitura do Gene Ribossômico/genética , Genoma Viral/genética , Humanos , Modelos Moleculares , Pandemias , SARS-CoV-2/fisiologia
5.
Nat Prod Res ; : 1-6, 2020 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-32400183

RESUMO

The objective was to investigate the role of consumption of grape juice (GJ), red wine (RW) or resveratrol solution (RS) on rats treated with a high-fat diet (HFD). Among the drinks offered, GJ had lower content of polyphenols and trans-resveratrol. Nevertheless, GJ showed similar content of anthocyanin and antioxidant activity to RW, although higher than RS. In rats treated with HFD, consumption of GJ presented best antioxidant and anti-inflammatory activity, reducing glutathione peroxidase and interleukin-6 serum levels. In addition, GJ promoted better levels of cholesterol and liver markers. On the other hand, RW aggravated the oxidizing effect of HFD, increasing catalase activity and interleukin-6 level. Already, RS showed no benefit in animals. Thus, GJ minimized the effects of HFD on oxidative stress and inflammation beyond promoted better levels of lipid profile and liver biomarkers. However, consumption of RS showed no benefit and RW revealed a pro-oxidant effect, as did HFD.

6.
Talanta ; 210: 120610, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31987179

RESUMO

In this work we propose the voltammetric analysis of contraceptive hormones ethinylestradiol (EE) and cyproterone acetate (CPA) using solid amalgam electrode fabricated with silver nanoparticles. To the best of our knowledge, this is the first report describing the simultaneous determination of these two compounds and also the first report of the use of amalgam electrode for analysis of EE and CPA. The voltammetric behavior of both substances was investigated by their reduction. An irreversible electrochemical process involving two protons and two electrons was found for each compound. The analytical assays were carried out using staircase voltammetry (SCV). Due to this, aiming to improve the analytical sensitivity, the cationic surfactant cetyltrimethylammonium bromide was also used. The instrumental and experimental parameters were studied and optimized to achieve the best conditions for the analysis. Under the optimum conditions, the voltammetric signals of EE and CPA showed dependence on the concentration range from 6.4 × 10-7 to 7.8 × 10-6 mol L-1 and from 1.0 × 10-6 to 1.0 × 10-5 mol L-1, respectively. The limits of detection obtained were 1.03 × 10-7 mol L-1 for EE and 2.99 × 10-7 mol L-1 for CPA. The analytical usefulness of the method was evaluated through its application on the simultaneous determination of EE and CPA in pharmaceutical formulations and urine samples. The two analytes were successfully quantified in these samples with good precision and the values found presented satisfactory concordance with the reference values, suggesting acceptable analytical efficiency for the approach described here.


Assuntos
Cetrimônio/química , Acetato de Ciproterona/análise , Técnicas Eletroquímicas , Etinilestradiol/análise , Tensoativos/química , Cátions/química , Composição de Medicamentos , Eletrodos , Humanos , Nanopartículas Metálicas/química , Conformação Molecular , Prata/química
8.
J Biomol NMR ; 69(1): 31-44, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28879611

RESUMO

Recently, 15N-detected multidimensional NMR experiments have been introduced for the investigation of proteins. Utilization of the slow transverse relaxation of nitrogen nuclei in a 15N-TROSY experiment allowed recording of high quality spectra for high molecular weight proteins, even in the absence of deuteration. Here, we demonstrate the applicability of three 15N-detected H-N correlation experiments (TROSY, BEST-TROSY and HSQC) to RNA. With the newly established 15N-detected BEST-TROSY experiment, which proves to be the most sensitive 15N-detected H-N correlation experiment, spectra for five RNA molecules ranging in size from 5 to 100 kDa were recorded. These spectra yielded high resolution in the 15N-dimension even for larger RNAs since the increase in line width with molecular weight is more pronounced in the 1H- than in the 15N-dimension. Further, we could experimentally validate the difference in relaxation behavior of imino groups in AU and GC base pairs. Additionally, we showed that 15N-detected experiments theoretically should benefit from sensitivity and resolution advantages at higher static fields but that the latter is obscured by exchange dynamics within the RNAs.


Assuntos
Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular/métodos , RNA/química
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