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

描述(申请人提供)：TB-RBP，也称为翻译蛋白，是一个27kD的 在时间上执行一系列重要生物功能的蛋白质 以及m RNA的空间表达。这种蛋白质结合了一个特定的区域，即Y和 H元件，在精子细胞发育过程中暂时沉默的选定的mRNAs。现在已知信使核糖核酸的结合是由鸟嘌呤控制的 核苷酸结合。Tb-RBP还可以作为马达蛋白的接头，以 促进发育中的雄性生殖细胞和神经元之间的mRNA运输 在大脑里。Tb-RBP还可以与细胞核中的单链DNA结合，并与 与白血病的DNA断裂和癌基因易位有关。我们建议的研究 应该会对信使核糖核酸识别的机制产生重要的见解， 沉默和运输，以及了解蛋白质是如何 促进致癌基因易位。 我们已经为TB-RBP或任何同系物制造了第一个X射线结构，来自一种 2.7地图。这种蛋白质展示了一个新的折叠，组装成一个八聚体 具有一个坚固的中央空腔，假设该空腔紧密地捆绑着 折叠的RNA元件。我们建议完成我们的模型，并将其改进为 衍射极限。我们还将GDP浸泡在我们的水晶中， 将蛋白质与GTP共结晶以分析构象变化 由GTP管理。我们建议通过以下方法来研究RNA和DNA的结合模式 与适当的核酸序列共结晶；这很可能 展示了一种新型的核酸结合。蛋白质Trax也已经被 表达。它是TB-RBP的同系物，并与TB-RBP形成多聚体，导致 它需要释放沉默的RNA。我们建议将天然单体结晶并 TB-RBPTRAX杂二聚体。此外，g-肌动蛋白和KIF3也是马达的一部分 已知的结合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。在协作中 与宾夕法尼亚大学医学院的一个小组一起，我们将制作和 鉴定关键的定点突变体以探索所有这些功能。目标是 是为了阐明TB-RBP组装、核酸结合、GTP的细节 效应器结合，以及与Trax和马达蛋白的相互作用。总而言之，这 工作应阐明分子结构和作用方式，并 一类重要的新的DNA/RNA结合蛋白的调控 引起科学界和医学界的兴趣。

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.