Mechanism of poly(A) polymerase processivity

描述（由申请人提供）：通过聚(A)聚合酶（Pap）对mRNA的3'端进行模板无关的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延伸作为分子手柄，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。我们最近研究的酵母聚(A)聚合酶（Pap1）与不可扩展ATP类似物3-dATP复合物的晶体结构揭示了碱基加成的催化机制，但没有揭示腺苷特异性的基础，也没有揭示聚(A)尾部穿过活性位点周围大间隙的路径，也没有揭示纯化后的Pap1在体外表现出高度加工性的结构基础。Pap1是理解聚合性的极好模型，因为与大多数其他聚合酶（包括哺乳动物的Pap）不同，它不需要额外的蛋白质来实现聚合性。我们建议使用x射线晶体学、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程序性延长mRNA的能力之间的关系。我们还提出了旨在从结构上理解Pap1是如何被mRNA切割/聚腺苷化复合体的另外两个组分Fip1和Yth1调节的工作。我们的初步晶体学结果为核苷酸特异性提供了基础。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。聚腺苷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.