PROTEIN RECOGNITION OF RNA MOTIFS: STRUCTURAL STUDIES

描述：这项工作的长期目标是确定原则 控制着蛋白质-RNA识别。蛋白质和RNA之间的相互作用 从调节基因表达到 指导细胞死亡。对这些原则的了解很少， 这在很大程度上是由于缺乏蛋白质-RNA复合物结构。这些 这些原理对于理解蛋白质-RNA机器很重要，例如 核糖体和剪接体，蛋白质-RNA结构和功能， 将军此外，它们对于发展知识型 针对RNA依赖性感染因子如HIV的治疗。 作为实现长期目标的一个步骤，本提案侧重于如何 核糖体失活蛋白局限曲菌素识别两种常见的基序（a tetraloop和一个G-凸起的交叉股A堆栈）的一个重要组成部分， 核糖体RNA值得注意的是，限制曲菌素， 与其功能同系物八叠球菌素，只切割一个磷酸二酯键， 在真核生物核糖体RNA中发现的大约7000个。这种强大的， 特异性毒素识别核糖体RNA的保守区域，称为 SRD是一种对蛋白质合成至关重要的结构域。 X射线晶体学、动力学和能量学研究将按顺序结合起来 为了更深入地了解限制曲肽如何识别两种基序， SRD RNA。第二个系列的晶体学研究将集中在突变体上 在SRD RNA中发现的两个基序中。这些研究将提供 对蛋白质识别原理的全新认识 这两个基序，因为没有相关的蛋白质-RNA复合物 结构. 局限曲菌素及其相关核糖体失活蛋白的临床研究进展 已经被它们在“神奇子弹”疗法中的潜在用途所证实， 通过将毒素与肿瘤特异性试剂连接， 抗体的确定RNA底物识别表面和接触 对限制作用至关重要的因素将有助于设计或选择 未来的核毒素疗法

DESCRIPTION: The long-term goal of this work is to determine the principles that govern protein-RNA recognition. Interaction between protein and RNA is central to biological processes ranging from regulating gene expression to directing cell mortality. Knowledge of these principles is poorly understood, due in large part to a paucity of protein-RNA complex structures. These principles are important for understanding protein-RNA machines, such as the ribosome and the spliceosome, and protein-RNA structure and function in general. In addition, they are important for developing knowledge-based therapies against RNA-dependent infectious agents such as HIV. As a step toward the long-term goal, this proposal focuses on how the ribosome-inactivating protein restrictocin recognizes two common motifs (a tetraloop and a G-bulged cross-strand A stack) in an essential piece of ribosomal RNA. Remarkably, restrictocin, which shares 86% sequence identity with its functional homolog sarcin, cleaves only one phosphodiester bond out of the approximately 7000 found in eukaryotic ribosomal RNA. This potent and specific toxin recognizes a conserved region of ribosomal RNA called the sarcin/ricin domain (SRD) that is essential for protein synthesis. X-ray crystallographic, kinetic and energetic studies will be combined in order to obtain a deeper understanding of how restrictocin recognizes both motifs in the SRD RNA. A second series of crystallographic studies will focus on mutants in the two motifs found in the SRD RNA. These studies will provide fundamentally new insights into the principles that govern protein recognition of these two motifs, because there is an absence of related protein-RNA complex structures. Clinical interest in restrictocin and related ribosome-inactivating proteins has been invigorated by their potential use in "magic bullet" therapies that direct toxins to tumor cells by linking them to tumor-specific agents such as antibodies. Determining the RNA substrate recognition surface and the contacts that are critical for restrictocin action will aid the design or selection of future ribotoxin-based therapies.