Mechanism of poly(A) polymerase processivity

描述(由申请人提供)：由聚(A)聚合酶(Pap)对mRNA3‘端的模板无关的延长几乎在所有生物体中都发生。在真核生物中，Pap添加的非编码的mRNA延伸片段作为分子把手，与核输出、翻译和mRNA降解机制相互作用，强烈影响着mRNA的稳定性和翻译效率。我们最近研究的酵母聚(A)聚合酶(PAP1)与不可扩展的ATP类似物3-dATP形成的复合体的晶体结构揭示了碱基加成的催化机制，但不是腺苷专一性的基础，也不是多聚(A)尾巴通过活性部位周围的大裂缝的路径，也不是纯化的PAP1在体外表现出高度加工能力的结构基础。PAP1是理解加工性的一个很好的模型，因为不像大多数其他聚合酶，包括哺乳动物的Pap，它不需要额外的蛋白质来实现加工性。我们建议使用X射线结晶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拉长mRNA的能力之间的关系。我们还建议开展工作，旨在从结构上了解Pap1是如何受到mRNA切割/聚腺苷酸化复合体的另外两个成分Fip1和Yth1的调控。我们的初步结晶学结果表明了核苷酸特异性的基础。此外，我们最近在高纯度的PAP1中发现了意想不到的核糖核酸酶活性。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与这一重要调控过程相关的分子事件的动态模型。

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.