【作者】 史诺；

【导师】 滕脉坤； 牛立文；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 核苷酸酶是一个非常重要的水解酶家族,它们能够将核苷单磷酸水解成核苷和磷酸根离子,与核苷酸激酶所参与的生物学过程互补,以保证细胞内核苷单磷酸库的平衡。作为一类生命基本活动所必需的酶,核苷酸酶参与了转录,翻译和信号转导等一系列的生物学过程中,从原核生物到真核生物体内都发现了这类酶的存在。从分类学的角度来说,核苷酸酶一般都属于HAD(Haloalkanoate dehalogenase)水解酶大家族,SDT1是一种酿酒酵母源的高度特异性的嘧啶核苷酸酶,参与到酵母的转录过程中。我们表达纯化了SDT1的全长和截去N端的蛋白,并解析了截去N端的蛋白的晶体结构。通过结构分析我们发现,SDT1由帽子结构域和核心结构域两部分组成,两个结构域共同形成了一个经典的α/βRossmann桶的折叠花样,已经解析的HAD水解酶大家族成员一般都采取这种折叠花样。而酶活中心也正是位于这两个结构域之间。进一步,通过浸泡底物的方法,得到了底物和SDT1的复合物结构。通过对复合物的结构解析,我们解释了SDT1的底物特异性的特点,对酶活的机制也有了更深的理解,为进一步酶抑制剂和嘧啶特异性的核苷酸酶抗性药物设计提供了结构基础。真核生物的转录是一个高度保守的生物学过程, RNA聚合酶II是整个转录过程中起中心角色的蛋白质分子。RNA聚合酶II通过与一系列的辅助因子相互作用,调节着转录的起始、延伸、暂停和终止。RNA聚合酶II相关蛋白1复合物PAF1(RNA Polymerase-Associated Factor 1)在转录的全过程中都结合在RNA聚合酶II上,发挥重要的作用。作为PAF1复合物最早被发现的两个组分之一,CDC73参与到许多转录相关的过程中:结合到RNA聚合酶II上,招募和激活组蛋白调节因子以及其它转录激活因子的信号转导。然而到目前为止,PAF1以及CDC73在转录过程中发挥作用的具体机制并不清楚。我们表达纯化了酿酒酵母源的CDC73全长蛋白和C端结构域片断,并解析了C端结构域的晶体结构。通过结构分析发现,CDC73蛋白的C端结构域折叠成一个类似于小G蛋白的结构花样。虽然在后续的实验中,我们无法检测到CDC73的GTPase活性,但为进一步探索CDC73和PAF1复合物的内在作用机理提供一些线索和结构基础。更多还原

【Abstract】 The 5’-nucleotidases catalyze the dephosphorylation of ribo- and deoxyribonucleoside monophosphates to the corresponding nucleosides. These enzymes can counteract the activity of nucleoside kinases and compete with other enzymes that consume the nucleoside monophosphates, which balance NTP and dNTP pools in cell. SDT1,first found as the deletion inhibitory factor of transcription elongation factor TFIIS in S. cerevisiae, suppresses the damage induced by deletion of TFIIS by overexpression. Here we report the 1.9? crystal structure of the yeast SDT1 complexed with phosphate and magnesium ions. We also present a 1.7 ? crystal structure of D61A variant complexed with the optimal substrate U5P (uridine 5’-monophosphate). The native SDT1 shows a classic HAD enzymes superfamily structure. The D61A variant structure enables us to characterize that the enzyme’s specificity for the ribo- form of nucleoside 5’-monophosphate is contributed to Asp-63, Asp-130 and Tyr-193.The yeast Paf1 complex (Paf1C), composed of the proteins Paf1, Cdc73, Ctr9, Leo1, and Rtf1, associates with RNA polymerase II (pol II) from the promoter to the 3′end formation site of mRNA encoding genes. As one component of the first two identified subunits of the Paf1 complex, Cdc73_Yeast (yCdc73) takes part in many transcription related processes including: binding to the RNA polymerase II; recruitment and activation of histone modification factors; communication with other transcriptional activators. The human homolog of yCdc73, also called as parafibromin, is identified as a tumor suppressor linked to breast, renal and gastric cancer. However, the functional mechanism of Cdc73 is not clear until recently. Here, we show the 2.1? crystal structure of the highly conserved C-terminal region of yCdc73. It reveals that Cdc73 appears as a GTPase-like fold. We hope that our found sheds new light on the function modes of cdc73 and Paf1 complex.更多还原