POLY(A) POLYMERASE AND FIP1 PEPTIDE COMPLEX

POLY(A) 聚合酶和 FIP1 肽复合物

• 批准号: 7602288
• 负责人: Alex ANDREW BOHM
• 金额: $ 0.39万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602288
• 关键词: 3' 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

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 在真核生物中，称为poly（a）尾巴的非编码mRNA扩展作为分子 与核出口，翻译和mRNA降解机械相互作用的手柄， 并强烈影响mRNA稳定性和翻译效率。这些尾巴是由多蛋白复合物（酵母中的〜14蛋白）形成的，该复合物识别出新生转录本的3'未翻译区域中的信号序列，并在由这些信号确定的位点上切割前体RNA。这种切割的前MRNA用作通过3'-End加工复合物的催化亚基添加聚（A）尾巴的底漆，Poly（a） - 聚合酶（酵母中的Pap1）。尽管孤立的Pap1酶在体外保留了野生型活性水平，但当与其在细胞内部构成束缚的复合物分离时，它并不能保留其RNA靶标或过程动力学的特异性。 FIP1是327个氨基酸的酸性蛋白，被认为是一种脚手架蛋白，酵母3'裂解/聚腺苷酸化合物的许多成分与聚（A）聚合酶相互作用。它是酵母裂解/聚腺苷酸化络合物的唯一组成部分，已显示与聚合酶直接相互作用。 FIP1与纳摩尔亲和力结合到PAP1，但经受凝胶过滤色谱法的速度异常大，并且被认为在pap1上和关闭pap1时都非常扩展。 FIP1的缺失研究表明，酵母生存能力是80至105之间的区域。为了更好地了解这些蛋白质的相互作用，我们在Pap1和Fip1区域之间形成了复合物的晶体。 凝胶电泳和质谱法证实了复合物的形成。 我们预计该综合体的结构将使我们能够开始定位其余的 相对于聚合酶的复合物。