Mechanism of poly(A) polymerase processivity

Poly(A) 聚合酶持续合成能力的机制

• 批准号: 6771700
• 负责人: Alex ANDREW BOHM
• 金额: $ 27.74万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6771700
• 关键词: DNA directed RNA polymerase; X ray crystallography; crosslink; enzyme activity; enzyme biosynthesis; enzyme mechanism; enzyme structure; fluorescence resonance energy transfer; messenger RNA; protein protein interaction; translation factor

DESCRIPTION (provided by applicant): Template-independent elongation of the 3' end of mRNA by poly(A) polymerase (Pap) occurs in virtually all organisms. In eukaryotes, the non-coding mRNA extensions added by Pap serve as molecular handles, which interact with nuclear export, translation and mRNA degradation machinery, and strongly effect mRNA stability and translational efficiency. Our recent crystal structure of yeast poly(A) polymerase (Pap1) in complex with the non-extendable ATP analog 3-dATP revealed the catalytic mechanism for base addition, but not the basis for adenosine specificity, the path of the poly(A) tail through the large cleft surrounding the active site, or the structural basis for the high degree of processivity exhibited in vitro by purified Pap1. Pap1 is an excellent model for understanding processivity, since unlike most other polymerases, including mammalian Pap, it does not require additional proteins to achieve processivity. We propose to use x-ray crystallography as well as fluorescence resonance energy transfer, cross-linking, and activity measurements to study the relationship between the structure of Pap1 and its ability to specifically and processively elongate mRNA. We also propose work aimed at a structural understanding of how Pap1 is regulated by two other components, Fip1 and Yth1, of the mRNA cleavage/polyadenylation complex. Our preliminary crystallographic results suggest a basis for nucleotide specificity. Also, we have very recently identified a unexpected RNase activity in highly purified Pap1. Polyadenylation is a major regulator of protein expression. The work we propose will provide a dynamic model of the molecular events associated with this important regulatory process.

描述（由申请人提供）： mRNA 3' 末端通过聚腺苷酸聚合酶（Pap）的模板独立延伸几乎发生在所有生物体中。在真核生物中，Pap 添加的非编码 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。我们最近的酵母聚腺苷酸聚合酶 (Pap1) 与不可延伸的 ATP 类似物 3-dATP 复合物的晶体结构揭示了碱基添加的催化机制，但不是腺苷特异性的基础，即聚腺苷酸的路径尾部穿过活性位点周围的大裂口，或者是纯化的 Pap1 在体外表现出高度持续性的结构基础。 Pap1 是理解持续合成能力的绝佳模型，因为与大多数其他聚合酶（包括哺乳动物 Pap）不同，它不需要额外的蛋白质来实现持续合成能力。我们建议使用 X 射线晶体学以及荧光共振能量转移、交联和活性测量来研究 Pap1 的结构与其特异性和持续延长 mRNA 的能力之间的关系。我们还提出了旨在从结构上理解 Pap1 如何受 mRNA 切割/多聚腺苷酸化复合物的另外两个成分 Fip1 和 Yth1 调节的工作。我们的初步晶体学结果表明了核苷酸特异性的基础。此外，我们最近在高度纯化的 Pap1 中发现了意想不到的 RNase 活性。多腺苷酸化是蛋白质表达的主要调节因子。我们提出的工作将提供与这一重要调控过程相关的分子事件的动态模型。