| Combining Gel Retardation and Footprinting to Determine Protein-DNA Interactions of Specific and/or Less Stable Complexes
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Author:
Date:
2020-12-05
[Abstract] DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to interpret if the binding of the protein to the DNA is weak, the protein has a fast off-rate, or if several different protein-DNA complexes are formed. Our protocol differs from traditional footprinting protocols, because it provides a method to isolate the protein-DNA complex from a native gel after treatment with the footprinting agent, thus removing the bound DNA from the free DNA or other protein-DNA complexes. The DNA is then extracted from the isolated complex before electrophoresis on a sequencing gel to determine the footprinting pattern. This analysis provides a possible solution for those who have been unable to use ...
[摘要] [摘要] DNA足迹是研究蛋白质-DNA相互作用的经典技术。但是，如果蛋白质与DNA的结合较弱，蛋白质的脱落速率较快，或者形成了几种不同的蛋白质-DNA复合物，则传统的足迹方案可能会失败或难以解释。我们的协议不同于传统的足迹协议，因为它提供了一种在使用足迹剂处理后从天然凝胶中分离蛋白质-DNA复合物的方法，从而从游离DNA或其他蛋白质-DNA复合物中去除了结合的DNA。然后从分离的复合物中提取DNA，然后在测序凝胶上电泳以确定印迹模式。该分析为无法使用传统足迹法确定蛋白质与DNA接触的人提供了可能的解决方案。
[背景]核酸酶/化学足迹是一个典型的方法来探测蛋白质-DNA相互作用（腊士和施米茨，1978;萨瑟-德怀特和Gralla，1989;汉普等人，2007） ...
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| Mapping mRNA-18S rRNA Contacts Within Translation Initation Complex by Means of Reverse Transcriptase Termination Sites and RNAseq
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Author:
Date:
2020-08-20
[Abstract] The nucleotides involved in RNA-RNA interaction can be tagged by chemical- or UV-induced crosslinking, and further identified by classical or modern high throughput techniques. The contacts of mRNA with 18S rRNA that occur along the mRNA channel of 40S subunit have been mapped by site-specific UV crosslinking followed by reverse transcriptase termination sites (RTTS) using radioactive or fluorescent oligonucleotides. However, the sensitivity of this technique is restricted to the detection of those fragments that resulted from the most frequent crosslinkings. Here, we combined RTTS with RNAseq to map the mRNA-18S rRNA contacts with a much deeper resolution. Although aimed to detect the interaction of mRNA with the ES6S region of 18S rRNA, this technique can also be applied to map the ...
[摘要] [摘要 ] 可以通过化学或紫外线诱导的交联来标记参与RNA-RNA相互作用的核苷酸，并通过经典或现代的高通量技术对其进行进一步鉴定。沿着40S亚基的mRNA通道发生的18S rRNA与mRNA的接触已通过位点特异性UV交联进行了定位，随后使用放射性或荧光寡核苷酸进行了逆转录酶终止位点（RTTS）。但是，该技术的敏感性仅限于检测由最频繁的交联产生的那些片段。在这里，我们将RTTS与RNAseq结合使用，以更深的分辨率绘制了mRNA-18S rRNA接触图。尽管旨在检测mRNA与18S rRNA的ES6S区域的相互作用，但该技术也可以用于绘制mRNA与其他非编码RNA分子的相互作用（在转录，剪接或RNA介导的转录后调控过程中（例如，snRNA，microRNA和lncRNA）。
[背景 ] 是与非编码RNA的mRNA的相互作用volved mRNA中的生命周期的每个步骤，从它的生物合成和处理进入细胞核至细胞质中的翻译和最终降解。这些相互作用可以在大分子机器核糖体和剪接体，以及在较小的复合物如RISC或者发生（RNA诱导的沉默复合物）或lncRNA介导的基因表达调节期间（皮萨列夫等人，2008; Engreitz 。等人，2014 Sharma ...
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