FUNCTIONAL STUDIES OF THE YEAST POLY(A) POLYMERASE

酵母多聚(A)聚合酶的功能研究

基本信息

批准号：
6525434
负责人：
CLAIRE L MOORE
金额：
$ 40.92万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-09-01 至 2004-08-31
项目状态：
已结题

项目摘要

Polyadenylation is essential for mRNA synthesis and its efficient translation. Regulation of this process can also affect the amount and type of mRNA derived from a gene, and thus becomes part of the cell's repertoire of responses to stimuli governing growth and differentiation. The enzyme poly(A) polymerase (PAP) is responsible for adding adenosines to the mRNA 3' end. For this activity, it needs binding sites to bring its substrates, ATP and an RNA strand, into proximity at its catalytic center. PAP must also have domains to mediate association with specificity factors which guide it to the appropriate 3' ends and modulate its activity so that it processively synthesizes tails of correct length. The mammalian PAP is further regulated by post-translational modification. The purpose of this study is to understand the molecular mechanism of PAP function, using a combination of biochemical, biophysical, and genetic approaches, and the Saccharomyces cerevisiae PAP, Pap1, as our model. First, we will use mutagenesis and kinetic analysis to explore the functional importance of motifs in Pap1 which are similar to ones found in RNases, RNA binding proteins, and other polymerases. This approach will be complemented by structural studies using X0ray diffraction analysis of crystals formed from purified Pap1 alone and in complex with ATP and RNA primer. We will also characterize the interactions of Pap1 with subunits of the PF I polyadenylation factor and explore the mechanisms by which these interactions affect Pap1 activity. Finally, we will ask what cellular factors are responsible for the novel ubiquitination of Pap1 in the G2 portion of the cell cycle and determine how this modification affects Pap1 function. Understanding the organization of important domains in a simple polymerase such as PAP can give insights into how functional motifs were combined or modified through evolution to yield the current spectrum of polymerases and nucleic acid modifying enzymes. The structural and mutational analyses will help us understand how PAP selects its substrates, how it differentiates between substrate and product, and how it advances along the single-stranded poly(A) chains as it synthesis progresses. Knowledge of the function and organization of PAP's enzymatic domains is necessary to understand the consequences of modification and protein/protein interactions which modulate PAP function. Information gained about yeast specificity factors will help us to define evolutionary relationships between polyadenylation factors in yeast and higher eukaryotes and to discern why interactions between some factors have changed while others are more highly conserved.

聚腺苷酸化对于mRNA合成及其有效翻译至关重要。该过程的调节也会影响从基因中得出的mRNA的数量和类型，因此成为细胞对刺激的刺激响应的曲目的一部分。酶聚（A）聚合酶（PAP）负责将腺苷添加到mRNA 3'端。对于此活动，它需要结合位点将其底物，ATP和RNA链带入其催化中心的接近度。 PAP还必须具有与特异性因子相关的域，这些特异性因素将其引导到适当的3'末端并调节其活动，以便其过程合成正确的长度。哺乳动物PAP受翻译后修饰进一步调节。这项研究的目的是使用生化，生物物理和遗传方法以及酿酒酵母Pap1的糖疗法的结合来理解PAP功能的分子机制，作为我们的模型。首先，我们将使用诱变和动力学分析来探索PAP1中基序的功能重要性，这些基序与RNase，RNA结合蛋白和其他聚合酶相似。结构研究将使用X0AR衍射分析对单独纯化的Pap1形成并与ATP和RNA底漆复杂化的晶体进行互补。我们还将表征Pap1与PF I聚腺苷酸化因子亚基的相互作用，并探讨这些相互作用影响Pap1活性的机制。最后，我们将询问哪些细胞因子是导致Pap1在细胞周期的G2部分中的新型泛素化的原因，并确定这种修饰如何影响Pap1功能。了解简单聚合酶中重要结构域（例如PAP）中的组织可以深入了解如何通过进化将功能性基序组合或修饰，以产生当前的聚合酶和核酸修饰酶的光谱。结构和突变分析将有助于我们了解PAP选择其底物，如何区分底物和产品，以及它在合成过程中如何沿单链poly（a）链的进步。必须了解PAP的酶促结构域的功能和组织，以了解调节PAP功能的修饰和蛋白质/蛋白质相互作用的后果。获得有关酵母特异性因素的信息将有助于我们定义酵母中聚腺苷酸化因子和更高真核生物之间的进化关系，并辨别为什么某些因素之间的相互作用发生了变化，而其他因素之间的相互作用是更高度保守的。