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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在真核生物中，被称为聚(A)尾的非编码mRNA延伸作为分子 手柄与核出口、翻译和信使核糖核酸降解机制相互作用， 并且强烈地影响了mRNA的稳定性和翻译效率。这些尾巴是由一个多蛋白复合体(酵母中约14个蛋白质)形成的，它识别新生转录本3‘非翻译区的信号序列，并在这些信号决定的位置切割前体RNA。这一裂解的前-mRNA作为引物，通过3‘-末端加工复合体的催化亚单位聚(A)-聚合酶(酵母中的PAP1)加成了聚(A)尾巴。虽然分离的PAP1酶在体外保持了野生型的活性水平，但当它与细胞内其结构性连接的复合体的其余部分分离时，它不能保持其RNA靶标或过程动力学的特异性。 Fip1是一种由327个氨基酸组成的酸性蛋白质，被认为是一种支架蛋白，酵母3‘裂解/多腺苷基化复合体的许多成分通过它与聚(A)聚合酶相互作用。它是酵母裂解/聚腺苷酸化复合体中唯一已被证明与聚合酶直接相互作用的成分。FIP1与PAP1具有纳摩尔亲和力，但在凝胶过滤层析中运行异常大，被认为在PAP1上和离开PAP1时都非常延长。对Fip1的缺失研究表明，在80到105之间的区域是酵母存活所必需的。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的这个区域之间的复合体的晶体。 凝胶电泳法和质谱仪证实了络合物的形成。我们预计，建筑群的结构将使我们能够开始定位其余部分 关于聚合酶的复合体。