Mechanism of poly(A) polymerase processivity

描述（由申请人提供）：几乎所有生物体中均存在多聚腺苷酸聚合酶（Pap）引起的mRNA 3'末端的模板非依赖性延长。在真核生物中，Pap所添加的非编码mRNA延伸作为分子把手，与核输出、翻译和mRNA降解机制相互作用，强烈影响mRNA的稳定性和翻译效率。我们最近的酵母多聚腺苷酸聚合酶（Pap 1）与不可延伸的ATP类似物3-dATP复合的晶体结构揭示了碱基加成的催化机制，但不是腺苷特异性的基础，多聚腺苷酸尾通过活性位点周围的大裂缝的路径，或纯化的Pap 1在体外表现出的高度持续合成能力的结构基础。Pap 1是理解持续合成能力的一个很好的模型，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Pap 1的结构与其特异性和procancelongate mRNA的能力之间的关系。我们还提出了工作的目的是在结构上的理解，如何Pap 1是由其他两个组件，Fip 1和Yth 1，mRNA切割/多聚腺苷酸化复合物的调节。我们初步的晶体学结果表明核苷酸特异性的基础。此外，我们最近在高度纯化的Pap 1中发现了一种意想不到的RNA酶活性。多聚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一个动态模型的分子事件与这一重要的调控过程。

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.