Site-Directed Chemical Probing to map transient RNA/protein interactions
Site-Directed Chemical Probing to map transient RNA/protein interactions
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DOI：
10.1016/j.ymeth.2016.12.011
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发表时间：
2017-03-15
期刊：
影响因子：
4.8
通讯作者：
Marzi, Stefano
中科院分区：
文献类型：
--
作者：
Duval, Melodie;Marenna, Alessandra;Marzi, Stefano
RNA-蛋白质相互作用是许多生物过程的基础，形成紧密和稳定的功能性核糖核蛋白（RNP）复合物（即核糖体）或短暂的复合物，如涉及RNA伴侣蛋白的复合物。为了定位蛋白质与RNA分子相互作用的位点，一种常见的简单而廉价的生物化学方法是足迹技术。该蛋白质在RNA上留下其足迹，作为保护相互作用区域免受化学修饰或用化学或酶促核酸酶获得的切割的屏障。该方法已被证明是有效的，在体外研究的组织稳定的RNA-蛋白质复合物。然而，当蛋白质以高解离速率非常动态地结合RNA时，通常难以观察到保护作用。对于这些瞬态复合物的分析，我们描述了一种替代策略，适用于现场定向化学探测（SDCP）的方法，我们比较它与经典的足迹。SDCP依赖于RNA结合蛋白的修饰，以将RNA探针（通常为Fe-EDTA）拴系到特定的蛋白质位置。相互作用区域上的局部切割可用于定位蛋白质并将其结构域定位在RNA分子上。这种方法已被用于在过去监测稳定的复合物，我们在这里提供了一个详细的协议和一个实际的例子，其应用于大肠杆菌RNA伴侣蛋白S1及其与mRNA的过渡复合物的研究。(C)2017爱思唯尔公司All rights reserved.
RNA-protein interactions are at the bases of many biological processes, forming either tight and stable functional ribonucleoprotein (RNP) complexes (i.e. the ribosome) or transitory ones, such as the complexes involving RNA chaperone proteins. To localize the sites where a protein interacts on an RNA molecule, a common simple and inexpensive biochemical method is the footprinting technique. The protein leaves its footprint on the RNA acting as a shield to protect the regions of interaction from chemical modification or cleavages obtained with chemical or enzymatic nucleases. This method has proven its efficiency to study in vitro the organization of stable RNA-protein complexes. Nevertheless, when the protein binds the RNA very dynamically, with high off-rates, protections are very often difficult to observe. For the analysis of these transient complexes, we describe an alternative strategy adapted from the Site Directed Chemical Probing (SDCP) approach and we compare it with classical footprinting. SDCP relies on the modification of the RNA binding protein to tether an RNA probe (usually Fe-EDTA) to specific protein positions. Local cleavages on the regions of interaction can be used to localize the protein and position its domains on the RNA molecule. This method has been used in the past to monitor stable complexes; we provide here a detailed protocol and a practical example of its application to the study of Escherichia coli RNA chaperone protein S1 and its transitory complexes with mRNAs. (C) 2017 Elsevier Inc. All rights reserved.