Wilhelm J E, Vale R D
Department of Pharmacology, University of California, San Francisco 94143, USA.
Genes Cells. 1996 Mar;1(3):317-23. doi: 10.1046/j.1365-2443.1996.25026.x.
mRNA translation, stability, and localization are controlled by regulatory proteins that bind to specific RNA motifs. Since biochemical isolation of such proteins has often proven to be difficult, a genetic system for studying RNA-protein interactions would be of great utility in the identification of novel RNA binding proteins and in understanding how these proteins recognize particular RNA sequences. The bacteriophage lambda gene product N protein is a sequence specific RNA binding protein that when bound to its target sequence allows RNA polymerase to ignore transcription termination signals. The fact that the binding of N protein to RNA is directly coupled to gene expression suggests that N protein could be used to develop a general system for studying RNA-protein interactions.
Our results show that fusion of the RNA binding protein, R17, to N causes antitermination in a beta-galactosidase reporter construct that has the R17 binding site substituted for the normal N target sequence. This system can both detect low affinity interactions as well as discriminate between binding events with equilibrium dissociation constants from 10(-5) to 10(-8)M. The differences in antitermination activity with various mutant binding sites can be reliably detected by colony colour on X-Gal plates as well as by liquid culture assay.
We have demonstrated that N protein can cause antitermination through a heterologous RNA-protein interaction and that the system is capable of detecting RNA-protein interactions of differing affinities. This approach may also be useful in screening libraries for proteins that bind to novel RNA regulatory elements. Our results are also consistent with a model of N protein function in which binding to the nascent transcript increases the effective concentration of N in the vicinity of RNA polymerase leading to antitermination.
信使核糖核酸（mRNA）的翻译、稳定性及定位受与特定RNA基序结合的调控蛋白控制。由于此类蛋白的生化分离常常证明很困难，因此用于研究RNA-蛋白质相互作用的遗传系统在鉴定新型RNA结合蛋白以及理解这些蛋白如何识别特定RNA序列方面将具有极大的实用性。噬菌体λ基因产物N蛋白是一种序列特异性RNA结合蛋白，当其与靶序列结合时，能使RNA聚合酶忽略转录终止信号。N蛋白与RNA的结合直接与基因表达相关这一事实表明，N蛋白可用于开发一种研究RNA-蛋白质相互作用的通用系统。
我们的结果表明，将RNA结合蛋白R17与N融合，会在一个β-半乳糖苷酶报告构建体中导致抗终止，该构建体中R17结合位点取代了正常的N靶序列。该系统既能检测低亲和力相互作用，也能区分平衡解离常数在10⁻⁵至10⁻⁸M之间的结合事件。通过X-Gal平板上的菌落颜色以及液体培养测定，能够可靠地检测出不同突变结合位点的抗终止活性差异。
我们已经证明，N蛋白可通过异源RNA-蛋白质相互作用导致抗终止，并且该系统能够检测不同亲和力的RNA-蛋白质相互作用。这种方法在筛选与新型RNA调控元件结合的蛋白文库方面可能也很有用。我们的结果也与N蛋白功能模型一致，即与新生转录本的结合会增加RNA聚合酶附近N的有效浓度，从而导致抗终止。
