Department of Biochemistry and Molecular Biology, College of Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA.
RNA. 2010 Feb;16(2):253-8. doi: 10.1261/rna.1880410. Epub 2009 Dec 14.
The yeast three-hybrid system (Y3H) is a powerful tool to select or confirm RNA-protein interactions. Target protein recognition of an RNA insert within a test transcript depends on at least three factors: intrinsic protein affinity for the properly folded insert, retention of RNA insert tertiary structure within a longer RNA transcript, and accessibility of the RNA insert to the target protein. Y3H reporter gene readout reflects the combination of these factors. Here, we discuss RNA insert tertiary structure and accessibility in the Y3H as "RNA display." We review evidence that RNA display can sometimes be optimized during Y3H selections that do not increase the intrinsic affinity of an RNA insert for a target protein. This situation is more likely when a library of RNA inserts and heterogeneous flanking sequences is subjected to selection, and is less likely when point mutations are targeted to the insert in a fixed context. An RNA display vector with enhanced modularity has been developed to minimize sequence context effects in the Y3H.
酵母三杂交系统 (Y3H) 是一种强大的工具，可用于选择或确认 RNA-蛋白质相互作用。靶蛋白对测试转录本中 RNA 插入物的识别至少取决于三个因素：蛋白质与正确折叠的插入物的固有亲和力、较长 RNA 转录本中 RNA 插入物三级结构的保留以及 RNA 插入物对靶蛋白的可及性。Y3H 报告基因读数反映了这些因素的组合。在这里，我们将 Y3H 中的 RNA 插入物三级结构和可及性称为“RNA 展示”。我们回顾了证据，表明在不增加 RNA 插入物与靶蛋白固有亲和力的情况下，有时可以在 Y3H 选择中优化 RNA 展示。当对 RNA 插入物和异质侧翼序列的文库进行选择时，这种情况更有可能发生，而当在固定背景下针对插入物进行点突变时，这种情况不太可能发生。已经开发了具有增强的模块化的 RNA 显示载体，以最大限度地减少 Y3H 中的序列背景效应。
