Department of Chemistry and Biochemistry, P.O. Box 951569, University of California, Los Angeles, CA 90095-1569, USA.
Structure. 2011 Jul 13;19(7):999-1010. doi: 10.1016/j.str.2011.03.022.
dsRBDs often bind dsRNAs with some specificity, yet the basis for this is poorly understood. Rnt1p, the major RNase III in Saccharomyces cerevisiae, cleaves RNA substrates containing hairpins capped by A/uGNN tetraloops, using its dsRBD to recognize a conserved tetraloop fold. However, the identification of a Rnt1p substrate with an AAGU tetraloop raised the question of whether Rnt1p binds to this noncanonical substrate differently than to A/uGNN tetraloops. The solution structure of Rnt1p dsRBD bound to an AAGU-capped hairpin reveals that the tetraloop undergoes a structural rearrangement upon binding to Rnt1p dsRBD to adopt a backbone conformation that is essentially the same as the AGAA tetraloop, and indicates that a conserved recognition mode is used for all Rnt1p substrates. Comparison of free and RNA-bound Rnt1p dsRBD reveals that tetraloop-specific binding requires a conformational change in helix α1. Our findings provide a unified model of binding site selection by this dsRBD.
dsRBDs 通常具有一定的特异性结合 dsRNAs,但这一基础的原理理解得还很差。Rnt1p 是酿酒酵母中的主要 RNase III,它使用其 dsRBD 识别保守的四环折叠结构,切割含有由 A/uGNN 四核苷酸环封闭的发夹的 RNA 底物。然而,Rnt1p 底物中 AAGU 四核苷酸环的鉴定提出了一个问题,即 Rnt1p 是否以不同于与 A/uGNN 四核苷酸环结合的方式结合这种非典型的底物。与 AAGU 封闭的发夹结合的 Rnt1p dsRBD 的溶液结构表明,四核苷酸环在与 Rnt1p dsRBD 结合时发生结构重排,采用与 AGAA 四核苷酸环基本相同的骨架构象,表明所有 Rnt1p 底物都使用保守的识别模式。对游离和 RNA 结合的 Rnt1p dsRBD 的比较表明,四核苷酸环特异性结合需要α1 螺旋的构象变化。我们的发现为该 dsRBD 结合位点选择提供了一个统一的模型。