Name: isolation of translating ribosomes containing peptidyl-trnas for functional and structural analyses
Uploaded: 2011-02-25T16:03:00
Duration: 11 min 19 s
Description: Watch this Scientific Journal Video about Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses at JoVE.com

LRC-V. хотел бы посвятить эту работу памяти доктора Adriel Д. Джонсон, прекрасным профессором которого приоритетом номер один всегда был воспитания студентов. Мы скучаем по тебе, Adriel. Мы благодарны Жаклин Морено за ее помощь в проведении экспериментов, описанных в данном исследовании. Это исследование было поддержано грантом предоставлена ​​CY от Национального научного фонда, MCB-0615390, а также запуск средства, предоставленные LRC-V. из Университета Алабамы в Хантсвилле.

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+translation+termination+at+the+upstream+open+reading+frame+in+s-adenosylmethionine+decarboxylase+mRNA.">Regulated translation termination at the upstream open reading frame in s-adenosylmethionine decarboxylase mRNA.</a> J. Biol. Chem. 277, 5988-5994 (2002).</li><li>Cruz-Vera, L. R., Rajagopal, S., Squires, C., Yanofsky, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+ribosome-peptidyl-tRNA+interactions+essential+for+tryptophan+induction+of+tna+operon+expression.">Features of ribosome-peptidyl-tRNA interactions essential for tryptophan induction of tna operon expression.</a> Mol. Cell. 19, 333-343 (2005).</li><li>Cruz-Vera, L. R., Yanofsky, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+Residues+Asp16+and+Pro24+of+tnaC-tRNAPro+Participate+in+Tryptophan+Induction+of+tna+Operon+Expression.">Conserved Residues Asp16 and Pro24 of tnaC-tRNAPro Participate in Tryptophan Induction of tna Operon Expression.</a> J. Bacteriol. 190, 4791-4797 (2008).</li><li>Cruz-Vera, L. R., Yang, R., Yanofsky, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tryptophan+inhibits+Proteus+vulgaris+tnaC+leader+peptide+elongation,+activating+tna+operon+expression.">Tryptophan inhibits Proteus vulgaris tnaC leader peptide elongation, activating tna operon expression.</a> J Bacteriol. 191, 7001-7006 (2009).</li><li>Cruz-Vera, L. R., New, A., Squires, C., Yanofsky, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomal+features+essential+for+tna+operon+induction:+tryptophan+binding+at+the+peptidyl+transferase+center.">Ribosomal features essential for tna operon induction: tryptophan binding at the peptidyl transferase center.</a> J. Bacteriol. 189, 3140-3146 (2007).</li><li>Shimizu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+translation+reconstituted+with+purified+components.">Cell-free translation reconstituted with purified components.</a> Nat. Biotechnol. 19, 751-755 (2001).</li><li>Kinoshita, Y., Nishigaki, K., Husimi, Y. F. l. u. o. r. e. s. c. e. n. c. e. -. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=isotope-+or+biotin-labeling+of+the+5+'-end+of+single-stranded+DNA/RNA+using+T4+RNA+ligase.">isotope- or biotin-labeling of the 5 '-end of single-stranded DNA/RNA using T4 RNA ligase.</a> Nucleic Acids Res. 25, 3747-3748 (1997).</li><li>Nishigaki, K., Taguchi, K., Kinoshita, Y., Aita, T., Husimi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Y-ligation:+an+efficient+method+for+ligating+single-stranded+DNAs+and+RNAs+with+T4+RNA+ligase.">Y-ligation: an efficient method for ligating single-stranded DNAs and RNAs with T4 RNA ligase.</a> Mol Divers. 4, 187-190 (1998).</li></ol>

Нет конфликта интересов объявлены.

Эта изоляция процедуре, описанной в настоящем отчете, высокой воспроизводимостью. К сожалению, наличие коротких пептидил-тРНК производится из той же последовательности перевода не может быть удобно избегать таких пептидил-тРНК может соответствовать 15-20% от общего объема материала изолированы (рис. 2С). Эти загрязнители могут увеличение концентрации при более высоких концентрациях меченных биотином мРНК были использованы или когда бесклеточных экстрактов используются старые или были повторно использованы несколько раз. Поэтому, очень важно для контроля качества и концентрации компонентов в реакциях пробирке перевода. Кроме того, коммерческие высокой эффективности восстановленного бесклеточных экстрактов доступны, которые могут использоваться для тех же целей (PURE системы) 18. Используя этот метод, биотин-16-UMP случайно включили по длине целевой мРНК. Существует возможность того, что некоторые ORF, содержащей уридин путей, возможно, включили это модифицированного нуклеотида в их последовательности, влияющие на их перевод. Переменная относительные концентрации biotin-16-UTP/UTP должны быть проверены в процессе синтеза мРНК с целью получения наиболее эффективного перевода. Альтернативные методы маркировки биотина может быть использован, ориентированных на 5&#39;-конце, наиболее стабильным концу мРНК. (Я), биотина может быть присоединен к 5&#39;-концу мРНК с использованием 3&#39;-NH2ATP (3&#39;-амино-, 3&#39;-дезоксиаденозина трифосфат) и Т4 РНК-лигазы 19. (II) 5&#39;-конце биотин-меченых олигонуклеотидных может быть присоединен к 5&#39;-концу мРНК с использованием РНК-лигазы Т4, а 20. (III) 3&#39;-конце меченных биотином олигодезоксинуклеотиды которые соответствуют комплементарную последовательность на 5&#39;-конце мРНК, также может быть использован для выделения переводе комплексов. Эти биотин помечены олигодезоксинуклеотиды может быть присоединен к SMB до изоляции. Позже малого и среднего бизнеса придается меченных биотином олигодезоксинуклеотиды может быть смешан с переводом смеси реакции, чтобы их взаимодействие с комплементарными последовательностями мРНК. После изоляции, гибридных РНК-ДНК регионе - и в регионе гибридизации между меченных биотином oligodeoxynucleotide и мРНК последовательности - может быть снижено использованием РНКазы H, отделяя застопорился комплексов из стрептавидином шарики. Этот альтернативный метод может позволить комплексов на работу в будущем структурный анализ с использованием выше методы разрешения.

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<td>Tris base</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>252859</td>
<td>&nbsp;</td>
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<td>Magnesium acetate</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>63049</td>
<td>&nbsp;</td>
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<td>Potassium glutamate</td>
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<td>G1501</td>
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<td>Ammonium acetate</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>09688</td>
<td>&nbsp;</td>
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<td>PMSF</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7626</td>
<td>&nbsp;</td>
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<td>Protein A sepharose 4B slurry beads</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9424</td>
<td>&nbsp;</td>
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<td>Leupeptin inhibitor</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L9783</td>
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<td>P7002</td>
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<td>85558</td>
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<td>D5758</td>
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<td>Tricine</td>
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<td>Sigma</td>
<td>T5816</td>
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<td>magnetic separation stands</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>Z5342</td>
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isolation of translating ribosomes containing peptidyl-trnas for functional and structural analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.

Watch this Scientific Journal Video about Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses at JoVE.com

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Главным препятствием для биохимического анализа рибосом содержащих зарождающиеся пептидил-тРНК была присутствии других рибосом в тех же образцах, рибосомы не участвует в переводе определенной последовательности мРНК анализируются. Мы разработали простую методологию, чтобы очистить, исключительно, содержащей рибосомы зарождающейся пептидил-тРНК интересов.

Выделение Перевод Рибосомы содержащих пептидил-тРНК на функциональные и структурные анализы

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