Structural analysis of TB-RBP, a DNA/RNA-binding protein
Structural analysis of TB-RBP, a DNA/RNA-binding protein
批准号:
6467700
负责人:
JON D Robertus
金额:
$27.76万
依托单位国家:
美国
项目类别:
财政年份:
2002
资助国家:
美国
项目状态:
已结题
起止时间:
2002-05-01 至 2006-04-30
关键词:
DNA DNA binding protein X ray crystallography actins chromatography computer simulation conformation crystallization disulfide bond guanine nucleotides intermolecular interaction messenger RNA model design /development molecular assembly /self assembly molecular site nucleoproteins physical model protein folding protein purification protein structure function site directed mutagenesis structural biology
中文摘要
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。
英文摘要
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.
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