Name: isolation of ribosome bound nascent polypeptides in vitro to identify translational pause sites along mrna
Uploaded: 2012-07-06T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA at JoVE.com

Эта работа финансировалась по программе по науке пограничной грант RGP0024.

1. Подготовка шаблона ДНК и транскрипции в пробирке <ol><li> Гена, клонируют в Т7 и / или, например, SP6 транскрипционный промотор. </li><li> Ибо в пробирке транскрипции ДНК-матрицы линеаризуется с соответствующим ферментом ограничение резки ниже по течению от кодона ORF остановки и / или ...

<ol>
	<li>Komar, A. 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+pause+for+thought+along+the+co-translational+folding+pathway.">A pause for thought along the co-translational folding pathway.</a> Trends Biochem. Sci. 34, 16-24 (2009).</li><li>Sharp, P. M., Cowe, E., Higgins, D. G., Shields, 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=Codon+usage+patterns+in+Escherichia+coli,+Bacillus+subtilis,+Saccharomyces+cerevisiae,+Schizosaccharomyces+pombe,+Drosophila+melanogaster+and+Homo+sapiens;+a+review+of+the+considerable+within-species+diversity.">Codon usage patterns in Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens; a review of the considerable within-species diversity.</a> Nucleic Acids Res. 16, 8207-8210 (1988).</li><li>Dittmar, K. A., Goodenbour, J. M., Pan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+differences+in+human+transfer+RNA+expression.">Tissue-specific differences in human transfer RNA expression.</a> PLoS Genet. 2, e221 (2006).</li><li>Ikemura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Codon+usage+and+tRNA+content+in+unicellular+and+multicellular+organisms.">Codon usage and tRNA content in unicellular and multicellular organisms.</a> Mol. Biol. Evol. 2, 13-34 (1985).</li><li>Wolin, S. L., Walter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+pausing+and+stacking+during+translation+of+a+eukaryotic+mRNA.">Ribosome pausing and stacking during translation of a eukaryotic mRNA.</a> EMBO J. 7, 3559-3569 (1998).</li><li>Krasheninnikov, I. A., Komar, A. A., Adzhubei, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonuniform+size+distribution+of+nascent+globin+peptides,+evidence+for+pause+localization+sites,+and+a+cotranslational+protein-folding+model.">Nonuniform size distribution of nascent globin peptides, evidence for pause localization sites, and a cotranslational protein-folding model.</a> J. Protein Chem. 10, 445-454 (1991).</li><li>Komar, A. A., Lesnik, T., Reiss, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synonymous+codon+substitutions+affect+ribosome+traffic+and+protein+folding+during+in+vitro+translation.">Synonymous codon substitutions affect ribosome traffic and protein folding during in vitro translation.</a> FEBS Lett. 462, 387-391 (1999).</li><li>Komar, A. A., Jaenicke, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+translation+of+&#947;+B+crystallin+and+its+circularly+permutated+variant+in+an+in+vitro+cell-free+system:+possible+relations+to+codon+distribution+and+protein+folding.">Kinetics of translation of &#947; B crystallin and its circularly permutated variant in an in vitro cell-free system: possible relations to codon distribution and protein folding.</a> FEBS Lett. 376, 195-198 (1995).</li><li>Jha, S., Komar, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birth,+life+and+death+of+nascent+polypeptide+chains.">Birth, life and death of nascent polypeptide chains.</a> Biotechnol. J. 6, 623-640 (2011).</li><li>Thanaraj, T. A., Argos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome-mediated+translational+pause+and+protein+domain+organization.">Ribosome-mediated translational pause and protein domain organization.</a> Protein Sci. 5, 1594-1612 (1996).</li><li>Kimchi-Sarfaty, C., Oh, J. M., Kim, I. W., Sauna, Z. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+"silent"+polymorphism+in+the+MDR1+gene+changes+substrate+specificity.">A "silent" polymorphism in the MDR1 gene changes substrate specificity.</a> Science. 315, 525-528 (2007).</li><li>Sch&#228;gger, H., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricine-sodium+dodecyl+sulfate-polyacrylamide+gel+electrophoresis+for+the+separation+of+proteins+in+the+range+from+1+to+100+kDa.">Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.</a> Anal. Biochem. 166, 368-379 (1987).</li><li>Shirole, N., Balasubramanian, S., Yanofsky, C., Cruz-Vera, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Translating+Ribosomes+Containing+Peptidyl-tRNAs+for+Functional+and+Structural+Analyses.">Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses.</a> J. Vis. Exp. (48), e2498 (2011).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. S., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324, 218-223 (2009).</li></ol>

Для воспроизводимости результатов, качества и концентрации компонентов, используемых для транскрипции в пробирке и перевод реакции являются критическими. В настоящем исследовании мы использовали коммерчески доступных наборов и экстракты, которые обеспечивают высокую воспроиз...

<table border="1"><tbody><tr><td> Название реагента / Kit </td><td> Компания </td><td> Номер по каталогу </td></tr><tr><td> MEGAscript T7 Высокая доходность Kit Транскрипция </td><td> Ambion </td><td> AM1333 </td></tr><tr><td> Ингибитор рибонуклеазы </td><td> Invitrogen </td><td> 15518012 </td></tr><tr><td> Транс [35 S]-Label </td><td> MP Biomedicals </td><td> 0151006 </td></tr><tr><td> Рибонуклеазы- </td><td> Invitrogen </td><td> 12091 </td></tr><tr><td> Кролик ретикулоцитов лизата система, нуклеазы Обработанные </td><td> Promega </td><td> L4960 </td></tr><tr><td> Кишечная палочка S30 экстракт системы линейных шаблонов </td><td> Promega </td><td> L1030 </td></tr><tr><td> Центрифугирование </td><td> Beckman Coulter </td><td> Optima TLX ультрацентрифуге </td></tr><tr><td> Хранение фосфор авторадиограммау </td><td> GE Healthcare </td><td> Тайфун 9410 изображений в режиме переменной </td></tr><tr><td> Программное обеспечение для рождающегося полипептида анализ </td><td> GE Healthcare </td><td> Изображение Квант TL, v2005 </td></tr></tbody></table>

isolation of ribosome bound nascent polypeptides in vitro to identify translational pause sites along mrna

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Watch this Scientific Journal Video about Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA at JoVE.com

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Техника для определения поступательного сайтов паузу на мРНК описано. Эта процедура основана на выделении зарождающейся полипептидов накопления на рибосомы в процессе искусственного перевода целевой мРНК, а затем размер анализ зарождающейся цепи использованием денатурирующих гель-электрофореза.

Выделение Рибосома связанных стадии становления Полипептиды<em&gt; В пробирке</em&gt;, Чтобы определить Поступательное сайтов Пауза Наряду мРНК

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Research

JoVE Journal

Biology

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

PAR-Clip - метод идентификации транскриптома всей места связывания РНК-связывающие белки

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

В пробирке транскрипции и укупорки мРНК люциферазы Gaussia Вслед за Трансфекция Сотовые HeLa

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Использование МЭСМ последовательности арест как средство изолировать Рибосома связанных полипептидов

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

Экстракорпоральное синтеза модифицированных мРНК для индукции экспрессии белка в клетках человека

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Измерение окислительного стрессоустойчивость<em&gt; Caenorhabditis Элеганс</em&gt; В микротитровальных пластинках 96-а

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Экспертиза белков, связанных с ДНК находятся в стадии становления в клетках млекопитающих, используя BrdU-чип-Slot-Западная Техника

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Измерение Жесткость<em&gt; Ex Vivo</em&gt; Аорте при помощи мыши атомно-силовой микроскопии

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Протокол для использования гена набор обогащения анализ для выявления соответствующих животных модель для трансляционного исследования

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

В пробирке SUMOylation Assay для изучения сумо E3 лигаза активность

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Быстрая фиксация in vivo и выделение трансляционных комплексов из эукариотических клеток

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...