Investigation of mRNA quadruplex formation in Escherichia coli
Investigation of mRNA quadruplex formation in Escherichia coli
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DOI：
10.1038/nprot.2009.111
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发表时间：
2009-11
期刊：
影响因子：
14.8
通讯作者：
Markus Wieland;J. Hartig
中科院分区：
文献类型：
--
作者：
Markus Wieland;J. Hartig
本文提出的方案允许通过将基因表达水平与相应结构的体外稳定性相关联来研究细菌5′-非翻译区（UTR）中不寻常核酸结构的形成。特别是，我们描述了引入G-四链体形成序列接近核糖体结合位点（RBS）的mRNA的报告基因和随后读出的表达水平。插入一个稳定的二级结构导致RBS的掩盖，并最终降低基因表达水平。引入序列的结构和稳定性进一步通过圆二色性（CD）光谱和热熔融实验表征。然后将抑制的程度与相应四链体结构的稳定性相关联，从而可以判断除了热力学稳定性之外的因素是否影响给定四链体序列在体内的形成。测量基因表达水平需要2天，包括克隆; CD实验每次实验需要5小时。
The protocol presented here allows for the investigation of the formation of unusual nucleic acid structures in the 5′-untranslated region (UTR) of bacteria by correlating gene expression levels to thein vitrostability of the respective structure. In particular, we describe the introduction of G-quadruplex forming sequences close to the ribosome-binding site (RBS) on the mRNA of a reporter gene and the subsequent read-out of the expression levels. Insertion of a stable secondary structure results in the cloaking of RBS and eventually reduced gene expression levels. The structures and stability of the introduced sequences are further characterized by circular dichroism (CD) spectroscopy and thermal melting experiments. The extent of inhibition is then correlated to the stability of the respective quadruplex structure, allowing judgement of whether factors other than thermodynamic stability affect the formation of a given quadruplex sequencein vivo. Measuring gene expression levels takes 2 d including cloning; CD experiments take 5 hours per experiment.