Name: protein-trna agarose gel retardation assays for the analysis of the n6-threonylcarbamoyladenosine tcda function
Uploaded: 2017-06-21T12:30:00.000+00:00
Duration: 8 min 3 s
Description: Watch this Scientific Journal Video about Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function at JoVE.com

MCV é membro do programa Intramural CIB &quot;Molecular Machines for Better Life&quot; (MACBET). A pesquisa que conduziu a esses resultados recebeu financiamento do Instituto Espanhol de Saúde Carlos III (PI12 / 01667 a MCV), o Ministério da Economia e Competitividade (CTQ2015-66206-C2-2-R e SAF2015-72961-EXP para MCV) ), O Governo Regional de Madri (S2010 / BD-2316 a MCV) e o Projeto ComplexINC (Contrato n.º 279039 a MCV) da Comissão Européia (Programa-Quadro 7 (FP7)). Os financiadores não tiveram papel no design do estudo, na coleta e análise de dados, na decisão de publicação ou na elaboração do manuscrito.

Cuidado: consulte todas as folhas relevantes de dados de segurança de material (MSDS) antes de usar. Vários dos produtos químicos utilizados neste protocolo são tóxicos e corrosivos. Use práticas adequadas ao realizar a extração de ARNt usando fenol, incluindo o uso de uma chaminé e equipamento de proteção pessoal (óculos de segurança, luvas, bata de laboratório, calças completas, sapatos fechados, etc. ). Este protocolo usa uma mancha de ácido nucleico não tóxico. O uso de manchas alternativas pode exigir precauções adicionais e disposição especializada do agente de coloração se for tóxico e / ou cancerígeno ( por exemplo , brometo de etidio). 1. Preparação do Gel de Agarose <ol><li> Pesar 2 g de agarose e adicioná-lo a uma garrafa de vidro para preparar um gel de agarose a 2% p / v. </li><li> Adicione 100 mL de tampão de transição de ácido tris-borato-etilenodiaminotetraacético (EDTA) (TBE) ao frasco contendo agarose. Derreta-se por aquecimento em um microondas. Com intervalos de 30 s, arraste o conteúdo para misturarBem e repita este passo até que a agarose esteja completamente dissolvida. Deixe a solução de agarose arrefecer até aproximadamente 50-60 ° C. </li><li> Tape as bordas abertas de uma bandeja de gel para criar um molde, coloque um pente apropriado para criar os poços e coloque a agarose fundida nele. Evite a formação de bolhas e deixe-o solidificar à temperatura ambiente. </li><li> Uma vez solidificado, coloque a bandeja de gel em uma unidade de eletroforese e preenchê-lo com tampão TBE até o gel estar completamente coberto. </li></ol> 2. Preparação do ARNt <ol><li> Expresso tRNA (UUU) em E. coli <ol><li> Na parte da manhã, use um frasco de E. coli BL21 (DE3) congelado transformado com o vetor de expressão pET23d-EcUUU 2 (abriga o gene que codifica o tRNA de E. coli (UUU)), para criar um Luria Broth (LB) de baixo teor de sal, Placa suplementada com 100 μg / mL de ampicilina. Inverta a placa e coloque-a em uma incubadora de 37 ° até que as colônias apareçam (tarde da tardeOon). </li><li> Inocular uma colônia única bem cultivada em 5 mL de LB mais 100 μg / mL de ampicilina e crescer durante a noite a 220 rpm a 37 ° C. </li><li> Na manhã seguinte, elimine as células (6.500 xg por 10 minutos a 4 ° C), substitua o meio com 5 mL de LB fresco mais 100 μg / mL de ampicilina e ressuspenda. Use esta suspensão para inocular 200 ml de LB mais 100 μg / mL de ampicilina. Cresça durante 5 h a 220 rpm a 37 ° C. </li><li> Quando a densidade óptica da cultura a 590 nm (OD 590 ) atingiu 0,6 - 0,8, adicione 1 mM de IPTG à cultura para desencadear a expressão do gene tRNA (UUU). Incubar a 37 ° C durante 3 h. </li><li> Colher as células por centrifugação da cultura a 6.500 xg durante 10 min a 4 ° C. </li></ol></li><li> Lise e extração <ol><li> Ressuspender o sedimento celular em 5 mL de tampão de lise (acetato de sódio 0,3 M, EDTA 10 mM). A partir deste ponto, usam buffers autoclavados e mantenham todas as amostras contendo tRNA a 4 ° C para evitar que tDegradação de ARN. </li><li> Adicione 1 volume de fenol ácido (pH 4,3) ao sedimento de células ressuspensas na exaustão. Misture durante 1 min por inversão para lisar as células e centrifugar a 10 000 xg (10 min, 4 ° C). </li><li> Recolher a fase aquosa superior que contém o tRNA solúvel (UUU) (e menores concentrações dos outros grupos de ARNt) usando uma pipeta com o cuidado de não aspirar a fase orgânica (inferior). Descarte a camada inferior e o grânulo, que consiste na fase orgânica e nos detritos celulares, num recipiente adequado. </li><li> Precipite o tRNA misturando completamente a fase solúvel (aquosa) com 2,5 volumes de etanol a 100% v / v por inversão durante 1 h a 4 ° C. Centrifugar a mistura a 15 000 xg (30 min, 4 ° C). </li><li> Decantar cuidadosamente o sobrenadante e ressuspender o sedimento rico em ARNt em 4 mL de tampão de filtração em gel (fosfato de sódio 20 mM ou fosfato de potássio, pH 7,4, EDTA 0,1 mM). Execute 5 μL do conjunto de ARNt ressuspenso em um gel de agarose a 2% p / v. NOTA:O ARNt de boa qualidade aparece como uma banda única (tamanho molecular entre 50-100 pb). </li></ol></li><li> Purificação do tRNA (UUU) <ol><li> Seguindo a prática cromatográfica padrão 12 , carregue o tRNA ressuspenso (UUU) em uma coluna de filtração de gel preparativa de alta resolução (dimensões: 16 x 600 mm, volume do leito: 120 mL, intervalo de resolução: 1.000-70.000 Da) previamente equilibrado em tampão de filtração em gel (na fase móvel). Defina o caudal para 1 mL / min para eluir as amostras. NOTA: O pico de ARNt (UUU) elui aproximadamente a 75,5 mL. Tipicamente, pode ser obtido um rendimento de 0,5-1,0 mg / L de tRNA de cultura. </li><li> Analisar as amostras em volumes de eluição representativos por eletroforese de 5 a 10 μL de cada amostra em um gel de agarose a 2% p / v. </li></ol></li></ol> 3. Preparação da amostra <ol><li> Expresse e purifique TcdA de acordo com López-Estepa et al . 2 . NOTA: Até 250 μMTcdA pode ser usado para o estudo. </li><li> Pipetar a quantidade correspondente de TcdA conforme a Tabela 1 em tubos de centrífuga de 1,5 mL devidamente rotulados. Adicionar 1,6 mM tris (2-carboxietil) fosfina (TCEP) (concentração final) e misture suavemente com uma pipeta. Feche a tampa e incube a mistura à temperatura ambiente durante 5 min. </li><li> Adicionar 10 μM de adenosina trifosfato (ATP) e 50 mM de MgCl2 (concentrações finais). Misturar com uma pipeta e incubar a mistura durante 5 minutos à temperatura ambiente. </li><li> Adicione tRNA purificado (UUU) (seção 2.3) de acordo com a Tabela 1 , misture com uma pipeta e incube à temperatura ambiente durante 5 min. </li><li> Adicione solução salina tamponada com fosfato (PBS) até 100 μL de volume final. Misturar adequadamente e adicionar glicerol até uma concentração final de 10% v / v. </li><li> Vortex a amostra e centrifugue brevemente o conteúdo do tubo (10 000 xg, 1 min, 4 ° C). </li></ol> 4. Desenvolvimento de carregamento de gel e eletroforese<ol><li> Coloque a unidade de eletroforese em uma bandeja cheia de gelo triturado para manter a câmara a 4 ° C durante a corrida de eletroforese. </li><li> Com cuidado, pipetar 5 μL de cada amostra no poço correspondente. Adicione um marcador de tamanho de DNA adequado. </li><li> Conecte os eletrodos nas ranhuras correspondentes da fonte de alimentação. Ligue a fonte de alimentação e ajuste a tensão para 100 V e execute o gel por 90 min. </li></ol> 5. Coloração de gel para observar a interação de proteína de ARNt <ol><li> Prepare a solução de coloração misturando 50 mL de TBE com 1x de uma mancha de DNA adequada. Evite expor a solução de coloração à luz solar cobrindo o recipiente de coloração com papel alumínio. NOTA: As manchas de DNA geralmente são armazenadas como ações concentradas de 10.000 ou 20.000x. </li><li> Após 90 min de eletroforese, retire cuidadosamente o gel da bandeja e coloque-o em um recipiente com solução de coloração. Coloque-o em um balancim a baixa velocidade de balanço na temperatura ambienteUre por 30 min. </li><li> Retire o gel de agarose do recipiente de coloração, aperte-o cuidadosamente em um pedaço de tecido de celulose ou papel de filtro para remover o excesso de líquido e coloque-o diretamente em um transiluminador. Ligue a luz UV para visualizar bandas contendo tRNA. Pegue uma fotografia do gel. </li><li> Coloração de proteínas (opcional). <ol><li> Descarte a solução de coloração de DNA de forma segura (de acordo com os procedimentos de segurança) e substitua-a por solução de 50 ml de Blue Brilliant Blue (CBB). Coloque a bandeja em um balancim à temperatura ambiente e coloque durante a noite (&gt; 12 h). Descarte a solução de coloração CBB e substitua-a por 50 mL de PBS para remover o excesso de corante. Destain durante 1 h em um rocker. </li></ol></li><li> Visualize os complexos de ARNt que contêm proteínas sob um transiluminador leve e tirar fotografias do gel para comparar com a imagem de ARN tRNA UV. </li></ol>

<ol>
	<li>Scott, V., Clark, A. R., Docherty, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gel+retardation+assay.">The gel retardation assay.</a> Methods Mol Biol. 31, 339-347 (1994).</li><li>L&#243;pez-Estepa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crystal+structure+and+small-angle+X-ray+analysis+of+CsdL/TcdA+reveal+a+new+tRNA+binding+motif+in+the+MoeB/E1+superfamily.">The crystal structure and small-angle X-ray analysis of CsdL/TcdA reveal a new tRNA binding motif in the MoeB/E1 superfamily.</a> Plos One. 10 (4), e0118606 (2015).</li><li>Miyauchi, K., Kimura, S., Suzuki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cyclic+form+of+N6-threonylcarbamoyladenosine+as+a+widely+distributed+tRNA+hypermodification.">A cyclic form of N6-threonylcarbamoyladenosine as a widely distributed tRNA hypermodification.</a> Nat Chem Biol. 9 (2), 105-111 (2013).</li><li>Bolstad, H. M., Botelho, D. J., Wood, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+protein-protein+interactions+within+the+Cysteine+Sulfinate+Desulfinase+Fe-S+cluster+biogenesis+system.">Proteomic analysis of protein-protein interactions within the Cysteine Sulfinate Desulfinase Fe-S cluster biogenesis system.</a> J Proteome Res. 9 (10), 5358-5369 (2010).</li><li>Loiseau, L., Ollagnier-de Choudens, S., Lascoux, D., Forest, E., Fontecave, M., Barras, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+heteromeric+CsdA-CsdE+cysteine+desulfurase,+assisting+Fe-S+cluster+biogenesis+in+Escherichia+coli.">Analysis of the heteromeric CsdA-CsdE cysteine desulfurase, assisting Fe-S cluster biogenesis in Escherichia coli.</a> J Biol Chem. 280 (29), 26760-26769 (2005).</li><li>Fried, M. G., Crothers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equilibrium+studies+of+the+cyclic+AMP+receptor+protein-DNA+interaction.">Equilibrium studies of the cyclic AMP receptor protein-DNA interaction.</a> J Mol Biol. 172 (3), 241-262 (1984).</li><li>Garner, M. M., Revzin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gel+electrophoresis+method+for+quantifying+the+binding+of+proteins+to+specific+DNA+regions:+application+to+components+of+the+Escherichia+coli+lactose+operon+regulatory+system.">A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system.</a> Nucleic Acids Res. 9 (13), 3047-3060 (1981).</li><li>Rio, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+mobility+shift+assays+for+RNA-protein+complexes.">Electrophoretic mobility shift assays for RNA-protein complexes.</a> Cold Spring Harb Protoc. 2014 (4), 435-440 (2014).</li><li>Ryder, S. P., Recht, M. I., Williamson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+protein-RNA+interactions+by+gel+mobility+shift.">Quantitative analysis of protein-RNA interactions by gel mobility shift.</a> Methods Mol Biol. 488, 99-115 (2008).</li><li>Yakhnin, A. V., Yakhnin, H., Babitzke, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel+mobility+shift+assays+to+detect+protein-RNA+interactions.">Gel mobility shift assays to detect protein-RNA interactions.</a> Methods Mol Biol. 905, 201-211 (2012).</li><li>Ream, J. A., Lewis, L. K., Lewis, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+agarose+gel+electrophoretic+mobility+shift+assay+for+quantitating+protein:+RNA+interactions.">Rapid agarose gel electrophoretic mobility shift assay for quantitating protein: RNA interactions.</a> Anal Biochem. 511, 36-41 (2016).</li><li>Hagel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel-filtration+chromatography.">Gel-filtration chromatography.</a> Curr Protoc Mol Biol. , (2001).</li></ol>

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O teste de retardamento de gel de proteína-ARNt de agarose aqui descrito pode ser modificado de várias maneiras. Primeiro, a porcentagem de agarose no gel pode ser reduzida para permitir a separação e visualização de complexos proteína-ARNt significativamente maiores que os analisados ​​aqui (120 kDa). Em segundo lugar, se a proteína alvo for termolábil, devem ser tomadas precauções adicionais para garantir que a temperatura seja mantida abaixo da temperatura máxima aceitável, movendo o aparelho de elet...

<html> O teste de retardamento de gel 1 (também conhecido como ensaio de mudança de mobilidade eletroforética, EMSA) é um método eletroforético projetado para estudar e caracterizar interações proteína-ácido nucleico. Ele permite a análise do DNA da proteína e das interações proteína-ARN e, em particular, as interações entre as proteínas de ligação de ARNt e ARNt, utilizando componentes purificados ou misturas complexas de proteínas ( por exemplo , lisados ​​celulares) ou ácidos nucleicos ( por exemplo , tRNA Piscinas). Nós aplicamos ensaios de retardamento de gel ao estudo da interação entre ARNt purificado (UUU) e TcdA, uma N6-teronilcarbamoiladenosina (t 6 A) desidratase, que cicliza a cadeia lateral de treonilcarbamoílo anexada a A37 no anel de tronco anticodon (ASL) De tRNAs para t 6 cíclico (ct 6 A) 2 , 3 . TcdA também é conhecido como CsdL 3 ,F &quot;&gt; 4. O gene tcdA / csdL forma um cluster com os genes csdA e csdE 5 , que codificam a dessulfurase de cisteína e o aceitador de enxofre do sistema de desulfinato de cisteína sulfinase (CSD) e é necessário para manter a função TcdA in vivo 3 . O raciocínio por trás dos ensaios de retardamento de gel é que, enquanto as moléculas de ácido nucleico livres migram rapidamente para a frente da acrilamida ou géis de agarose em virtude da sua grande carga negativa, a mobilidade eletroforética de complexos de proteína dos mesmos ácidos nucleicos é dramaticamente reduzida. A redução da mobilidade é visualizada como uma &quot;mudança&quot; nos complexos de ácido nucleico-proteína, que se resolvem como bandas discretas. O complexo de ácido nucleico e proteína positivamente carregado e maior apresenta uma maior mudança ou redução na mobilidade em um gel. Uma vez que a neutralização da carga do complexo é um fenômeno universalPara os complexos proteína-ácido nucleico, o teste de retardamento do gel pode ser aplicado a uma ampla gama de complexos e tipos de ácidos nucleicos. O método é simples, barato e pode ser realizado em laboratórios com equipamentos mínimos. Requer apenas pequenas quantidades de proteínas e ARNt. Esta é uma vantagem em relação às técnicas biofísicas alternativas, que geralmente consomem maiores quantidades de amostra. O teste de retardamento de gel tem sido amplamente utilizado para o estudo de fatores de transcrição. O ensaio tem sido utilizado para analisar a cinética de ligação, a força e a especificidade dos fatores de transcrição para muitas seqüências de DNA diferentes. Foi nesse campo que a EMSA foi desenvolvida pela primeira vez 6 , 7 . Os ensaios de retardamento de gel com moléculas de ARN 8 , 9 , 10 , 11 e tRNA também foram utilizados na pesquisa de ribossomosE analisar, como fazemos aqui, as enzimas que modificam os nucleósidos de tRNA pós-transcrição. Muitos parâmetros diferentes afetam o resultado de um teste de retardamento de gel, como temperatura, qualidade da proteína e amostra de tRNA, e a força da ligação. O planejamento cuidadoso ea execução do experimento são cruciais para a interpretação dos resultados do ácido nucleico livre e das espécies ligadas a proteínas. Aqui, usamos o gene sintético que codifica tRNA (UUU) clonado em um vetor de expressão cujo promotor não possui o local de ligação do ribossoma Shine-Dalgarno, levando à síntese de grandes quantidades do tRNA 2 . Este protocolo detalhado destina-se a fornecer um guia claro para realizar experiências de retardamento de gel de ARNt, evitando erros comuns.

<table><tbody><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL21(DE3)</td><td>Novagen</td><td>70235</td><td>DE3 lysogen contains T7 polymerase upon IPTG induction.</td></tr><tr><td>pET23d</td><td>Novagen</td><td>69748-3</td><td>pET23d confers resistance to ampicillin; T7 promoter.</td></tr><tr><td>LB Broth, Low Salt (Lennox L Broth), Granulated</td><td>Melford</td><td>GL1703</td><td>Conventional LB Broth, High Salt, can also be used.</td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>10419313</td><td></td></tr><tr><td>Incubator Shaker (Thermostated)</td><td>NBS</td><td>Excella E24&amp;nbsp;</td><td>Any shaker incubator with temperature control can be used.</td></tr><tr><td>Isopropyl-beta-D-thiogalactopyranoside (IPTG), &gt;=99%</td><td>Acros Organics</td><td>BP1755</td><td></td></tr><tr><td>Sodium acetate, anhydrous, &gt;99%</td><td>Melford</td><td>B4017</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA), Disodium salt, &gt;99%</td><td>Acros Organics</td><td>AC327205000</td><td>Harmful if inhaled.</td></tr><tr><td>Centrifuge (refrigerated)</td><td>Eppendorf</td><td>5430</td><td></td></tr><tr><td>Centrifuge (refrigerated) rotor</td><td>Eppendorf</td><td>5430/5430P</td><td></td></tr><tr><td>Centrifuge</td><td>Sorvall</td><td>RC5C</td><td></td></tr><tr><td>Centrifuge rotor</td><td>Sorvall</td><td>SLA-3000</td><td></td></tr><tr><td>Phenol solution, Saturated with 0.1 M citrate buffer, pH 4.3 &amp;plusmn; 0.2</td><td>Sigma-Aldrich</td><td>P4682</td><td>Acute toxicity (oral, dermal, inhalation), corrosive</td></tr><tr><td>Ethanol absolute for analysis, 100% v/v</td><td>Merck Millipore</td><td>100983</td><td></td></tr><tr><td>Sodium phosphate dibasic dihydrate, Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, &gt;=99%</td><td>Sigma-Aldrich</td><td>71643</td><td></td></tr><tr><td>Potassium dihydrogen phosphate, KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>48,730,250</td><td></td></tr><tr><td>HiLoad 16/60 Superdex 75&amp;nbsp;</td><td>GE Healthcare</td><td>28989333</td><td></td></tr><tr><td>Agarose</td><td>Melford</td><td>MB1200</td><td></td></tr><tr><td>Tris [Tris(hydroxymethyl) aminomethane HCl]</td><td>Melford</td><td>T1513</td><td></td></tr><tr><td>Boric Acid</td><td>Melford</td><td>B0503</td><td></td></tr><tr><td>Microwave</td><td>Haier</td><td>HDA-2070M</td><td></td></tr><tr><td>TCEP [Tris(2-carboxyethyl)phosphine hydrochloride]</td><td>Sigma-Aldrich</td><td>C4706</td><td>Corrosive to metals and skin.</td></tr><tr><td>ATP [Adenosine 5&amp;prime;-triphosphate disodium salt hydrate], Grade I, &gt;=99%</td><td>Sigma-Aldrich</td><td>A2383</td><td></td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;), anhydrous, &gt;=98%</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Sodium chloride (NaCl), &gt;99%</td><td>Fisher Bioreagents</td><td>BP358</td><td></td></tr><tr><td>Potassium chloride (KCl), &gt;99%</td><td>Melford</td><td>P0515</td><td></td></tr><tr><td>NZYDNA Ladder VI</td><td>NZYtech</td><td>MB8901</td><td></td></tr><tr><td>Power supply</td><td>Consort</td><td>EV261</td><td></td></tr><tr><td>Electrophoresis unit</td><td>Real Laboratory</td><td>RELSMINI10</td><td></td></tr><tr><td>Real Safe nucleic acid staining</td><td>Real Laboratory</td><td>RBMSAFE</td><td>20,000X stock</td></tr><tr><td>UV Transilluminator (G:Box/EF)</td><td>Syngene</td><td>2216498</td><td></td></tr><tr><td>Coomassie Brilliant Blue G</td><td>Sigma-Aldrich</td><td>B0770</td><td></td></tr><tr><td>Rocker (Gyro-Rocker)</td><td>Stuart</td><td>SSL3</td><td></td></tr><tr><td>Mixer Vortex&amp;nbsp;</td><td>Fisher brand</td><td>13214789</td><td></td></tr></tbody></table>

protein-trna agarose gel retardation assays for the analysis of the n6-threonylcarbamoyladenosine tcda function

Demonstramos métodos para a expressão e purificação de tRNA (UUU) em Escherichia coli e a análise por ensaios de retardamento de gel da ligação de tRNA (UUU) a TcdA, uma N6-teronilcarbamoil-videina (t 6 A) desidratase, que cicliza o treonilcarbamoílo Cadeia lateral anexada a A37 no circuito anticodon (ASL) de tRNAs para t 6 A cíclico (ct 6 A). A transcrição do gene sintético que codifica tRNA (UUU) é induzida em E. coli com β-D-1-tiogalactopiranósido isopropílico 1 mM (IPTG) e as células contendo tRNA são colhidas 24 h após a indução. A fração de ARN é purificada usando o método de extração de fenol ácido. O ARNt puro é obtido por cromatografia de filtração em gel que separa eficientemente as moléculas de tRNA de tamanho pequeno de ácidos nucleicos intactos ou fragmentados maiores. Para analisar a ligação TcdA ao tRNA (UUU), TcdA é misturado com tRNA (UUU) e separado em um gel de agarose nativo a 4 ° C.O tRNA livre (UUU) migra mais rápido, enquanto os complexos TcdA-tRNA (UUU) sofrem um retardo de mobilidade que pode ser observado após a coloração do gel. Demonstramos que TcdA é uma enzima de ligação de tRNA (UUU). Este ensaio de retardamento de gel pode ser usado para estudar mutantes de TcdA e os efeitos de aditivos e outras proteínas na ligação.

Grandes quantidades de ARNt (UUU) podem ser obtidas expressando o gene de ARNt em E. coli sob o controle de um forte promotor de T7 indutível. O tRNA expresso (UUU) se acumula no citoplasma e é enriquecido sobre o conjunto de tRNAs naturalmente abundantes. Um processo de purificação em duas etapas consistindo em um passo de captura / extração e um passo de filtração / polimento de gel foi utilizado para obter o ARNt da classe EMSA. O passo de captura / extração usa pH ...

Watch this Scientific Journal Video about Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function at JoVE.com

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

Um protocolo é apresentado para a produção de tRNA (UUU) e a análise de tRNA (UUU) em complexo com a enzima TcdA por ensaios de retardamento de gel de agarose.

Ensaios de Retardamento de Gel de Agarose Protein-tRNA para Análise do<em&gt; N</em<sup&gt; 6</sup&gt; -Treonilcarbamoyladenosina TcdA Function

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of ...

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Research

JoVE Journal

Biochemistry

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

9.8K visualizações.  Spanish National Research Council (CSIC).  Um protocolo é apresentado para a produção de tRNA (UUU) e a análise de tRNA (UUU) em complexo com a enzima TcdA por ensaios de retardamento de gel de agarose. 

Vídeo: Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme&#39;s active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

preparação e<em&gt; In Vivo</em&gt; O uso de uma sonda baseado em atividades para<em&gt; N</em&gt; Amidase Ácido -acylethanolamine

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

Bioquímica

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

Ensaio in vitro de metilação tRNA com a Entamoeba histolytica DNA e Dnmt2 tRNA metiltransferase (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

Imunologia e Infecção

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.

Human telomerase reverse transcriptase (TERT) synthesizes not only telomeric DNA but also double-stranded RNA through RNA-dependent RNA polymerase activity. Here, we describe a newly established assay to detect RNA-dependent RNA polymerase activity of endogenous TERT.

Detecção semi-quantitativa de atividade RNA-dependente do RNA polimerase humana Telomerase Transcriptase reversa proteína

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

Genética

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

Purificação eficiente e desenvolvimento de ensaio baseado no LC-MS/MS para dez-onze translocação-2 5-metilcitosina dioxigenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

Um ensaio In Vitro para detectar a atividade de Transferase de tRNA-isopentenilo

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

Teste anticorpo-livre para a análise da atividade do methyltransferase do RNA

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy.
Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action.
Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control.
The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide.
This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates.

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

Gallium(III) 5,10,15-(tris)pentafluorophenylcorrole and its freebase analogue exhibit low micromolar cell cytotoxicity. This manuscript describes an RNA transcription reaction, imaging RNA with an ethidium bromide-stained gel, and quantifying RNA with UV-Vis spectroscopy, in order to assess transcription inhibition by corroles and demonstrates a straightforward method of evaluating anticancer candidate properties.

Um<em&gt; In Vitro</em&gt; Ensaio Enzimático para Medir Inibição da transcrição pelo gálio (III) e H<sub&gt; 3</sub&gt; 5,10,15-tris (pentafluorofenil) corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

Bioengenharia

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

Nucleocápside Annealing-Mediated Eletroforese (NAME) ensaio permite a identificação rápida de HIV-1 Nucleocápside Inibidores

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

CD Spectroscopy to Study DNA-Protein Interactions

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.

The interaction of an ATP-dependent chromatin remodeler with a DNA ligand is described using CD spectroscopy. The induced conformational changes on a gene promoter analyzed by the peaks generated can be used to understand the mechanism of transcriptional regulation.

Espectroscopia de CD para estudar interações DNA-proteína

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

Ensaios de Retardamento de Gel de Agarose Protein-tRNA para Análise do

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