POLY(A) POLYMERASE AND FIP1 PEPTIDE COMPLEX

POLY(A) 聚合酶和 FIP1 肽复合物

基本信息

批准号：
7726221
负责人：
Alex ANDREW BOHM
金额：
$ 0.49万
依托单位：
BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-18 至 2009-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7726221
关键词：
3&apos Untranslated Regions Affinity Amino Acids Binding Catalytic Domain Cells Cleaved cell Complex Computer Retrieval of Information on Scientific Projects Database Enzymes Eukaryota Eukaryotic Cell Functional RNA Funding Gel Chromatography Genetic Translation Grant Heterogeneous Nuclear RNA In Vitro Institution Kinetics Mass Spectrum Analysis Messenger RNA Molecular Multiprotein Complexes Nuclear Export Peptide Signal Sequences Peptides Poly(A) Tail Polyadenylation Polymerase Polynucleotide Adenylyltransferase Positioning Attribute Precursor RNA Process Proteins RNA Research Research Personnel Resources Rest Running Scaffolding Protein Signal Transduction Site Source Specificity Structure Tail Thinking Transcript United States National Institutes of Health Yeasts gel electrophoresis mRNA Precursor mRNA Stability mRNA Transcript Degradation

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In eukaryotes, the non-coding mRNA extensions known as poly(A) tails serve as molecular handles, which interact with nuclear export, translation and mRNA degradation machinery, and strongly effect mRNA stability and translational efficiency. These tails are formed by a multiprotein complex (~14 proteins in yeast) which recognizes signal sequences in the 3' untranslated region of a nascent transcript and cleaves the precursor RNA at a site determined by these signals. This cleaved pre-mRNA serves as a primer for addition of the poly(A) tail by the catalytic subunit of the 3'-end-processing complex, poly(A)-polymerase (Pap1 in yeast). Though isolated Pap1 enzyme retains wild type levels of activity in vitro, it does not retain specificity for its RNA target or processive kinetics when separated from the rest of the complex to which it is constitutively tethered within the cell. Fip1, an acidic protein of 327 amino acids, is thought to be a scaffolding protein through which many of the components of the yeast 3' cleavage/polyadenylation complex interact with poly(A) polymerase. It is the only component of the yeast cleavage/polyadenylation complex which has been shown to interact directly with the polymerase. Fip1 binds with nanomolar affinity to Pap1, but runs abnormally large when subjected to gel filtration chromatography and is thought to be very extended both on and off Pap1. Deletion studies of Fip1 have shown that the region between 80 and 105 is necessary for yeast viability. In an effort to better understand the interaction of these proteins we've formed crystals of the complex between Pap1 and this region of Fip1. Complex formation has been confirmed by gel electrophoresis and by mass spectrometry. We anticipate that the structure of the complex will allow us to begin to position the rest of the complex with respect to the polymerase.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。子项目和研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以在其他 CRISP 条目中表示。列出的机构是对于中心来说，它不一定是研究者的机构。在真核生物中，被称为多聚 (A) 尾的非编码 mRNA 延伸充当分子手柄，与核输出、翻译和 mRNA 降解机制相互作用，并强烈影响 mRNA 稳定性和翻译效率。这些尾部由多蛋白复合物（酵母中约 14 种蛋白）形成，该复合物识别新生转录物 3' 非翻译区的信号序列，并在由这些信号确定的位点切割前体 RNA。这种切割的前 mRNA 可作为引物，通过 3' 末端加工复合物 Poly(A) 聚合酶（酵母中的 Pap1）的催化亚基添加 Poly(A) 尾。尽管分离的 Pap1 酶在体外保留了野生型活性水平，但当与细胞内组成型束缚的复合物的其余部分分离时，它不保留其 RNA 靶标或进行动力学的特异性。 Fip1 是一种由 327 个氨基酸组成的酸性蛋白，被认为是一种支架蛋白，酵母 3' 切割/聚腺苷酸化复合物的许多成分通过该蛋白与聚腺苷酸聚合酶相互作用。它是酵母裂解/聚腺苷酸化复合物的唯一成分，已被证明可以直接与聚合酶相互作用。 Fip1 以纳摩尔级亲和力与 Pap1 结合，但在进行凝胶过滤色谱时运行异常大，并且被认为在 Pap1 内外都延伸得非常大。 Fip1 的删除研究表明，80 到 105 之间的区域对于酵母活力是必需的。为了更好地了解这些蛋白质的相互作用，我们形成了 Pap1 和 Fip1 该区域之间的复合物晶体。复合物的形成已通过凝胶电泳和质谱法得到证实。我们预计综合体的结构将使我们能够开始定位其余部分复合物相对于聚合酶的变化。