Effects of pH and salt concentration on the siRNA binding activity of the RNA silencing suppressor protein p19
Effects of pH and salt concentration on the siRNA binding activity of the RNA silencing suppressor protein p19
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DOI：
10.1016/j.febslet.2007.05.064
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发表时间：
2007-06-26
期刊：
影响因子：
3.5
通讯作者：
Pezacki, John Paul
中科院分区：
文献类型：
--
作者：
Koukiekolo, Roger;Sagan, Selena M.;Pezacki, John Paul
RNA沉默途径是真核生物抗病毒免疫反应的重要组成部分，特别是在植物中。反过来，许多病毒已经进化出逃避或抑制这一途径的机制。像康乃馨意大利环斑病毒（CIRV）这样的tombusvirus表达一种19 kDa的蛋白（p19），该蛋白是受感染植物中RNA沉默的抑制因子。该蛋白作为二聚体，通过结合间隙中带电残基之间的静电相互作用特异性地与短干扰RNA （siRNA）结合。由于不同宿主的pH和盐浓度差异很大，我们研究了这些参数对CIRV p19 siRNA结合活性的影响。之前，我们利用Ni2+-NTA包被的96孔板建立了一种便捷的荧光检测CIRV p19:siRNA结合的方法。使用这种方法，我们观察到CIRV p19蛋白以纳摩尔亲和力与siRNA结合，并且这种结合对pH和盐浓度敏感。ph -解离常数谱显示CIRV p19:siRNA结合依赖于三种不同的表观pK(a)值。从曲线推断出的值分别为7.1、8.0和10.6，我们将其解释为一个或多个组氨酸、半胱氨酸和赖氨酸残基的电离。我们发现CIRV p19对RNA沉默的最佳抑制发生在pH为6.2至7.6的范围内。(c) 2007年欧洲生化学会联合会。Elsevier B.V.版权所有。
The RNA silencing pathway is an important component of the anti-viral immune response in eukaryotes, particularly in plants. In turn, many viruses have evolved mechanisms to evade or suppress this pathway. Tombusviruses such as the Carnation Italian ringspot virus (CIRV) express a 19 kDa protein (p19) that is a suppressor of RNA silencing in infected plants. This protein acts as a dimer and binds specifically to short-interfering RNA (siRNA) through electrostatic interactions between charged residues in the binding cleft. Since pH and salt concentrations can vary widely from host to host, we have investigated the influence of these parameters on the siRNA binding activity of CIRV p19. Previously, we established a convenient fluorescence-based method for assaying CIRV p19:siRNA binding using Ni2+-NTA coated 96-well plates. Using), this method, we observe that the CIRV p19 protein binds to siRNA with nanomolar affinity and that this binding is sensitive to pH and salt concentration. The pH-dissociation constant profile shows that CIRV p19:siRNA binding is dependent on three different apparent pK(a) values. The values extrapolated from the curve are 7.1, 8.0 and 10.6 that we interpret as the ionization of one or more histidine, cysteine and lysine residues, respectively. We find that the optimal suppression of RNA silencing by CIRV p19 occurs in the pH range from 6.2 to 7.6. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.