RNA-protein interactions in the yeast three-hybrid system: Affinity, sensitivity, and enhanced library screening
RNA-protein interactions in the yeast three-hybrid system: Affinity, sensitivity, and enhanced library screening
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DOI：
10.1261/rna.7202705
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发表时间：
2005-02-01
期刊：
影响因子：
4.5
通讯作者：
Wickens, M
中科院分区：
文献类型：
--
作者：
Hook, B;Bernstein, D;Wickens, M
酵母三杂交系统已成为分析rna -蛋白相互作用的有效工具。将RNA序列与与转录激活结构域（AD）相连的RNA结合蛋白结合进行测试。一个多产的rna -蛋白相互作用激活体内的报告基因。该系统已被用于测试候选RNA-蛋白对，在每个相互作用的伙伴中分离突变，并识别结合给定RNA序列的蛋白质。然而，报告基因激活与rna -蛋白相互作用的体外亲和力之间的关系尚未得到系统的研究。这限制了对数据的解释，并使新战略的制定复杂化。在这里，我们分析了三杂交系统的几个关键参数，以PUF蛋白FBF-1与一系列RNA靶点的相互作用为模型。我们比较了两个报告基因的激活作为相互作用的体外亲和力的功能。HIS3和LacZ的表达水平与10倍Kd范围内的亲和力直接相关。报告基因的表达也与活化结构域融合蛋白的丰度直接相关。我们描述了一个新的酵母菌株，YBZ1，简化了cDNA/AD文库的筛选。该菌株具有MS2外壳蛋白高亲和变体的串联，首尾二聚体，融合到LexA dna结合蛋白的单体上。我们发现，在cDNA文库筛选中使用该菌株增加了检测到的真实的、序列特异性阳性的数量，同时减少了假的、rna独立阳性的背景。
The yeast three-hybrid system has become a useful tool in analyzing RNA-protein interactions. An RNA sequence is tested in combination with an RNA-binding protein linked to a transcription activation domain (AD). A productive RNA-protein interaction activates a reporter gene in vivo. The system has been used to test candidate RNA-protein pairs, to isolate mutations in each interacting partner, and to identify proteins that bind a given RNA sequence. However, the relationship between reporter gene activation and in vitro affinity of an RNA-protein interaction has not been examined systematically. This limits interpretation of the data and complicates the development of new strategies. Here, we analyze several key parameters of the three-hybrid system, using as a model the interaction of a PUF protein, FBF-1, with a range of RNA targets. We compare activation of two reporter genes as a function of the in vitro affinity of the interaction. HIS3 and LacZ expression levels are directly related to affinity over a 10-fold range of Kd. Expression of the reporter genes also is directly related to the abundance of the activation domain fusion protein. We describe a new yeast strain, YBZ1, that simplifies screening of cDNA/AD libraries. This strain possesses a tandem, head-to-tail dimer of a high-affinity variant of MS2 coat protein, fused to a monomer of the LexA DNA-binding protein. We show that the use of this strain in cDNA library screens increases the number of genuine, sequence-specific positives detected, and at the same time reduces the background of false, RNA-independent positives.