STRUCTURE-FUNCTION ANALYSIS OF A SPLICEOSOMAL ATPASE

高等真核生物中的初级转录物通常含有间插 必须精确切除以产生功能性信使的序列 RNA。 因此，核前体mRNA剪接是调节 每个真核细胞的基因表达。 管制和替代 剪接在决定正常细胞发育和产生 高等真核生物中广泛的遗传多样性。 异常 剪接与某些疾病有关;例如， 剪接模式的改变与肿瘤的激活有关， c-Hras是人类癌症中最常见的突变基因之一。 虽然 在定义剪接的一般特征方面已经取得了很大进展 以及识别特定的组件，了解法规 这一过程将需要分析的拼接机制， 分子水平。 RNA-RNA和RNA-蛋白质相互作用的阐明 以及在细胞中实现构象变化的方式， spliceosome是这种理解的核心。 对这些问题的深入了解可以通过研究 ATP酶，如PRP 16的相互作用，已知在特定的功能， 拼接步骤。 该提案提出了实验，以描绘 蛋白质因子PRP 16利用ATP水解的分子机制 以促进剪接反应的最后步骤， 成熟RNA的形成。 PRP 16蛋白的功能结构域 将通过突变分析来确定。 这些研究很重要 通过将其冷冻在 不同的品脱（例如ATP结合，水解，结合或释放 剪接体）。被证明在某些功能上有缺陷的突变体 将为后续的遗传和生物化学研究提供有价值的工具， 研究，旨在分离新的剪接因子。 酵母的选择 作为一个系统，允许通用和生物化学的强大组合， 研究剪接中分子间相互作用的方法。 因为 剪接过程在进化上是保守的， 将对高等植物的基因表达产生重要影响。 真核生物 关于PRP 16功能机制的研究是特别重要的。 因为这种蛋白质似乎是一类 相关因素，DEAH盒家族成员。 这些事关 DEAD（asp-glu-ala-asp）盒蛋白的超家族， 它们是RNA依赖性ATP酶和RNA解旋酶。 是 一个令人着迷的可能性是，每一种剪接体DEAH蛋白， 像PRP 16一样，利用ATP水解促进蛋白质构象的变化。 剪接体在剪接中的一个独特步骤。 因此，对 PRP 16应该为理解他人的作用提供有价值的提示 家庭成员。

Primary transcripts in higher eukaryotes often contain intervening sequences which must be precisely excise to generate functional messenger RNAs. Nuclear pre-mRNA splicing is thus an essential step in regulating gene expression in every eukaryotic cell. Regulated and alternative splicing play a role in determining normal cell development and generate a broad spectrum of genetic diversity in higher eukaryotes. Aberrant splicing is associated with certain diseases;f or examples, disruption of splicing patterns has been implicated in the oncogenic activation of c-Hras, one of the most commonly mutated genes inhuman cancer. Although much progress has been made in defining the general features of splicing as well as identifying specific components, understanding the regulation of this process will require the analysis of the splicing machinery of the molecular level. Elucidation of RNA-RNA and RNA-protein interactions and the way in which conformational changes are achieved in the spliceosome is central to this understanding. Insights into these questions can be gained by studying the molecular interactions of ATPases, such as PRP16, known to function at specific steps of splicing. This proposal presents experiments to delineate the molecular mechanism by which the protein factor PRP16 uses ATP hydrolysis to promote the final steps of the splicing reaction leading to the formation of mature RNA. The functional domains of the PRP16 protein will be determined by mutational analysis. These studies are important to determine the PRP16 cycle in the splicing reaction by freezing it at distinct pints (e.g. ATP-binding, hydrolysis, binding to or release from the spliceosome). Mutants that prove to be defective in certain functions will provide valuable tools for subsequent genetic and biochemical studies, aimed at isolating novel splicing factors. The choice of yeast as a system permits the powerful combination of generic and biochemical approaches in studying the molecular interactions in splicing. Because the splicing process is evolutionary conserved, insights gained in yeast will have important implications for gene expression in higher eukaryotes. Studies concerning the mechanism of PRP16 function are of particular interest because this protein appears to be a paradigm for class of related factors, members of the DEAH box family. These are related to the superfamily of DEAD (asp-glu-ala-asp) box proteins, some members of which are demonstrated RNA-dependent ATPases and RNA helicases. It is a fascinating possibility that each of the spliceosomal DEAH proteins, like PRP16, uses ATP hydrolysis to promote a conformational change in the spliceosome at a distinct step in splicing. Thus, investigations of PRP16 should offer valuable hints for understanding the role of the other members of the family.