Structural analysis of TB-RBP, a DNA/RNA-binding protein

描述（由申请人提供）：TB-RBP，也称为translin，是一种27 kD的 在颞叶中执行一系列重要生物功能的蛋白质 和mRNA的空间表达。该蛋白质结合一个特定的区域，Y和 H元件，用于在精子细胞过程中暂时沉默的所选mRNA 发展现在已知mRNA的结合受鸟嘌呤控制 核苷酸结合TB-RBP还作为马达蛋白的接头， 促进mRNA在发育中的雄性生殖细胞之间的运输，并沿沿着神经元运输 在大脑里。TB-RBP还可以结合细胞核中的ssDNA，并已与 白血病中的DNA断裂和癌基因易位。我们提议的研究 应该会对mRNA识别机制产生重要的见解， 沉默和运输，以及了解蛋白质如何 促进致癌基因易位。 我们已经产生了TB-RBP或任何同系物的第一个X射线结构， 2.7张地图给这种蛋白质呈现出一种新的折叠方式，可以组装成八聚体 其具有一个基本的中心空腔， 折叠RNA元件。我们建议完成我们的模型，并将其细化到 衍射极限。我们还将GDP注入到我们的晶体中， 将蛋白质与GTP共结晶以分析构象变化 由GTP管理。我们建议检查模式的RNA和DNA的结合， 与适当的核酸序列共结晶;这很可能 表现出一种新型的核酸结合。TRAX蛋白也被 表达。它是TB-RBP的同系物，与TB-RBP形成多聚体， 释放沉默的RNA。我们建议将天然单体结晶， TB-RBPTRAX异二聚体。此外，g-肌动蛋白和KIF 3是马达的一部分， 已知结合TB-RBP的系统。共结晶和生物化学研究将 来定义这些分子机器是如何组装的。合作 与宾夕法尼亚大学医学院的一个小组合作，我们将 表征关键的定点突变体，以探索所有这些功能。目标 目的是阐明TB-RBP的组装、核酸结合、GTP 效应物结合以及与TRAX和马达蛋白的相互作用。在一起，这 工作应阐明分子结构和作用方式， 调节一类重要的新的DNA/RNA结合蛋白， 科学和医学界的兴趣。

DESCRIPTION (provided by applicant): TB-RBP, also called translin, is a 27 kD protein that performs a range of important biological functions in the temporal and spatial expression of mRNA. The protein binds a specific region, the Y and H elements, of selected mRNAs for temporary silencing during sperm cell development. Binding of mRNA is now known to be controlled by guanine nucleotide binding. TB-RBP also serves as an adapter to motor proteins to facilitate mRNA transport among developing male germ cells, and along neurons in the brain. TB-RBP can also bind ssDNA in the nucleus and has been associated with DNA breaks and oncogene translocation in leukemias. Our proposed research should produce important insights into the mechanism of mRNA recognition, silencing, and transport, as well as an understanding of how the protein facilitates oncogenic gene translocation. We have produced the first X-ray structure for TB-RBP, or any homologue, from a 2.7 A map. The protein, which exhibits a novel fold, assembles into an octamer with a substantial central cavity that is hypothesized to bind a compactly folded RNA element. We propose to complete our model and to refine it to the limits of diffraction. We also have soaked GDP into our crystals and co-crystallized the protein with GTP for analysis of the conformational changes governed by GTP. We propose to examine the mode of RNA and DNA binding by co-crystallization with appropriate nucleic acid sequence; this may well exhibit a novel type of nucleic acid binding. The protein TRAX has also been expressed. It is a homologue of TB-RBP and forms multimers with TB-RBP causing it to release silenced RNA. We propose to crystallize the native monomer and the TB-RBPTRAX heterodimer. In addition, g-actin and KIF3 are parts of motor systems known to bind TB-RBP. Co-crystallization and biochemical studies will be undertaken to define how these molecular machines assemble. In collaboration with a group at the University of Pennsylvania Medical School, we will make and characterize key site-directed mutants to explore all these functions. The goal is to elucidate the details of TB-RBP assembly, nucleic acid binding, GTP effector binding, and interactions with TRAX and motor proteins. Together, this work should elucidate the molecular structure and the mode of action and regulation of an important new class of DNA/RNA-binding proteins of great interest to the scientific and medical community.