Assays for eukaryotic translation factors that bind mRNA
Assays for eukaryotic translation factors that bind mRNA
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DOI：
10.1006/meth.1996.0431
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发表时间：
1997-04-01
期刊：
影响因子：
--
通讯作者：
Sonenberg, N
中科院分区：
文献类型：
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作者：
Merrick, WC;Sonenberg, N
在简要介绍了mRNA特异性翻译因子eIF4A、eIF4B和eIF4F的功能之后，本文提出了真核系统中与mRNA翻译激活相关的翻译因子的研究方法。从兔网织细胞裂解物中纯化eIF4A、eIF4B和eIF4F，同时纯化血红蛋白mRNA。这些纯化为RNA结合（如硝化纤维素过滤器上的保留）和RNA依赖的ATP水解的模型分析提供试剂。使用T7或SP6聚合酶作为RNA转录物的额外试剂，可以进行两种额外的检测，交联到mRNA的氧化帽或RNA解绕的atp依赖反应（解旋酶检测）。最后，描述了利用天然mrna的最具生物学意义的测定方法，即真正多肽链的合成，在本例中为血红蛋白。在本文处理生物测定的整个部分中，提供了一些有用的提示，以确保测定有效，并为对照实验提供了建议，以确保不研究人工制品。希望这些信息将有助于研究因子活性的调节（对于eIF4A， eIF4B或eIF4F）或各种mrna的翻译效率（有时受到调节）。(C) 1997学术出版社。
After a brief introduction to the function of the mRNA-specific translation factors eIF4A, eIF4B, and eIF4F, this article presents appropriate methodology for the study of the translation factors associated with the activation of mRNA for translation in eukaryotic systems. The purification of eIF4A, eIF4B, and eIF4F from rabbit reticulocyte lysates is given along with a procedure for the purification of hemoglobin mRNA. These purifications provide reagents for the model assays of RNA binding (as retention on nitrocellulose filters) and RNA-dependent ATP hydrolysis. With additional reagents available as RNA transcripts using either T7 or SP6 polymerase, two additional assays are possible, crosslinking to the oxidized cap of the mRNA or the ATP-dependent reaction of RNA unwinding (helicase assay). Finally, there is a description of the most biological assay for the utilization of natural mRNAs, the synthesis of the authentic polypeptide chain, in this instance hemoglobin. Throughout the portion of this article that deals with the biological assays, helpful hints are provided to ensure that the assay works, and suggestions are provided for control experiments to ensure that an artifact is not being studied. It is hoped that this information will facilitate the study of either the regulation of factor activity (for eIF4A, eIF4B, or eIF4F) or the translation efficiency (sometimes regulated) of various mRNAs. (C) 1997 Academic Press.