Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location
Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location
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DOI：
10.2144/000112243
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发表时间：
2006-09-01
期刊：
影响因子：
2.7
通讯作者：
Palmenberg, Ann C.
中科院分区：
文献类型：
--
作者：
Bochkov, Yury A.;Palmenberg, Ann C.
来自脑心肌炎病毒（EMCV）的内部核糖体进入位点（IRES）是广泛用于实验和药物应用的流行RNA元件，以在真核细胞或无细胞提取物中表达蛋白质。在单顺反子或双顺反子信使RNA（mRNA）中包含野生型元件赋予适当配置的顺反子高水平的帽非依赖性翻译活性。该元素的历史和商业IRES载体固有的序列衍生的实验结果不太为人所知。与双二糖报告构建体头对头比较，双顺反子构型的天然EMCV IRES比类似构型的pIRES载体引导8至10倍的蛋白质。它还产生了几乎两倍于pCITE（R）-1的蛋白质，pCITE（R）-1是一种早期的单顺反子迭代，在一个关键的结构基序中含有一个次优的A7序列。结果表明，研究者应该知道，并仔细报告他们的IRES在任何比较研究的顺序。说明了最佳活性的优选IRES（病毒碱基273-845）和最小IRES（病毒碱基400-836）。
The internal ribosomal entry site (IRES)from encephalomyocarditis virus (EMCV) is a popular RNA element used widely in experimental and pharmaceutical applications to express proteins in eukaryotic cells or cell-free extracts. Inclusion of the wild-type element in monocistronic or bicistronic messenger RNAs (mRNAs) confers a high level of cap-independent translation activity to appropriately configured cistrons. The history of this element and the experimental consequences of sequence derivations inherent to commercial IRES vectors are less well known. Compared head-to-head with dual-luciferose reporter constructs, a native EMCV IRES in a bicistronic configuration directed 8- to 10-fold more protein than a similarly configured pIRES vector. It also produced nearly twice as much protein as pCITE (R)-1, an early monocistronic iteration, harboring a suboptimal A7 sequence in a crucial structural motif. The results indicate that investigators should be aware of and carefully report the sequence of their IRES in any comparative study. The preferred IRES (viral bases 273-845) and the minimum IRES (viral bases 400-836)for optimum activity arc illustrated.