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

这项工作是由人类前沿科学计划授予RGP0024。

1。 DNA模板制备及体外转录<ol><li>感兴趣的基因被克隆在T7和/或如SP6的转录子。 </li><li> 在体外转录模板DNA线性适当的限制酶切割的ORF终止密码子和/或mRNA的3&#39;端下游。一个需要验证完整的线性质粒DNA的琼脂糖凝胶电泳上运行酶切产品。 </li><li>线性质粒用于体外转录反应。不同浓度的DNA模板可以进行测试，以确定最佳的DNA浓度在体外转录。一般来说，Ambion公司的MEGAscr...

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对于重复性的结果， 体外转录和翻译反应中使用的组件的质量和浓度是关键。在目前的研究中，我们已经使用市售的包和提取物，提供高度可重复性的数据，如果小心处理。然而，翻译能力的提取物可准备从细胞的选择，如果需要的话。质量可以影响mRNA的翻译，所以它是最重要的测试之前使用它在体外翻译的mRNA的完整性。 此外， 在体外翻译反应的时间?...

<table border="1"><tbody><tr><td> 试剂名称/盒 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> MEGAscript T7高产转录试剂盒</td><td> Ambion公司</td><td> AM1333 </td></tr><tr><td>核糖核酸酶抑制剂</td><td> Invitrogen公司</td><td> 15518012 </td></tr><tr><td>反式[35]标签</td><td> MP的Biomedicals </td><td> 0151006 </td></tr><tr><td>核糖核酸酶- </td><td> Invitrogen公司</td><td> 12091 </td></tr><tr><td>兔网织红细胞裂解系统，核酸治疗</td><td> Promega公司</td><td> L4960 </td></tr><tr><td> 大肠杆菌 S30提取物系统的线性模板</td><td> Promega公司</td><td> L1030 </td></tr><tr><td>离心</td><td>贝克曼</td><td>舰TLX超速离心机</td></tr><tr><td>存储磷放射自显影Ÿ </td><td> GE医疗集团</td><td>台风9410可变模式成像</td></tr><tr><td>新生多肽分析软件</td><td> GE医疗集团</td><td>图像定量TL，V2005 </td></tr></tbody></table>

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

翻译伸长率非均匀。 mRNA的二级结构，密码子的使用和mRNA的相关蛋白可能会改变核糖体的运动上的信息进行审查，见1。然而，它现在被广泛接受的同义密码子使用的非均匀平移伸长率的主要原因。同义密码子使用相同的频率。比其他2更频繁使用一些密码子的同义密码子的使用存在的偏见。密码子偏性是有机体以及组织特异性2,3。此外，密码子的使用频率是4同源tRNA的浓度成正比。因此，经常使用的密码子，将有相应的tRNA高出许多，这进一步意味着，频繁的密码子将被翻译速度比不经常。因此，将减缓地区稀有密码子（潜在暂停网站）丰富的mRNA作为一项规则上的信息核糖体运动GE和事业积累的新生肽5-8各自的大小。这些暂停的网站可以有功能的蛋白表达的影响， 审查的 mRNA稳定性和蛋白质折叠9。事实上，它表明，扶贫等暂停网站，可以改变的mRNA上的核糖体运动和随后可能会影响效率的翻译共同体内蛋白质折叠1,7,10,11。为了了解在体内的蛋白质折叠的过程中，在细胞中，蛋白质的合成过程，它必须获得全面的见解，在翻译延伸到沿mRNA的核糖体运动的影响密码子使用/ tRNA的内容最终加上。 在这里，我们描述一个简单的方法可以用来定位主要翻译为一个给定的mRNA翻译各种无细胞系统6-8暂停网站。这个程序是基于隔离accumulati新生多肽在体外的靶mRNA的翻译过程中核糖体纳克。理由是，在低频率的密码子，增加停留时间的增加相应大小的新生肽的核糖体结果。 在体外转录的mRNA存在放射性标记的氨基酸在体外翻译反应让新生链的检测。以孤立核糖体结合的新生多肽复合物的翻译反应顶端30％的甘油溶液离心分层。 polysom​​al颗粒新生多肽进一步用核糖核酸酶A和SDS-PAGE解决。这种技术可能可以被用于任何蛋白质，并允许沿mRNA的核糖体运动和检测的主要暂停网站分析。此外，该协议可以适应学习的因素和条件，可以改变核糖体的运动，从而可能还可以改变日é功能/蛋白质的构象。

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

描述的一种技术，以确定mRNA的翻译暂停网站。本程序是基于对新生多肽在体外培养的靶mRNA的翻译，其次是使用变性凝胶电泳新生链的大小分析核糖体积累的隔离。

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

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剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

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

科研

生物学

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

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

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

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

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

在体外合成在人体细胞修饰蛋白表达的mRNA的感应

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

测量氧化应激性<em&gt;秀丽隐杆线虫</em&gt;在96孔微量滴定板

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

考试蛋白在哺乳动物细胞中绑定到新生的DNA使用的BrdU芯片插槽西方技术

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

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

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

一个协议来使用基因集富集分析以确定合适的动物模型转化型研究

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

体外sumo 法研究相扑 E3 连接活动

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

从真核细胞 中快速体内 固定和分离转化复合物

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