Mechanisms of DNA and RNA transactions

项目摘要：该Mira提案整合并扩展了不同的调查思路 到目前为止，DNA和RNA交易得到了四项长期NIGMS赠款的支持。的目标是 这项研究的目的是：(一)了解执行核酸的酶的机制和结构 合成、修饰和修复；以及(Ii)阐明调节这些事件的因素。该项目 综合不同的实验方法(微生物学、生物化学、结构生物学、遗传学)和 将它们应用于从病毒到细菌再到真菌的各种模型系统。主要的主题是： (1)多核苷酸连接酶和信使核糖核酸末端识别的化学机制和结构基础 通过共价酶催化核苷酸转移到5‘-磷酸化末端的封端酶-(赖氨酰- Nζ)-NMP中间体。我们将解决典型的依赖于ATP的DNA连接酶和封顶的结构 酶作为其步骤1米氏与NTP和金属辅因子形成络合物。我们将澄清 NHEJ连接酶LigD为3‘-单核苷酸缺口，封闭酶为ppRNA。我们将利用时间- 错误的结晶学探讨金属在连接酶合成磷酸二酯中的作用。 (2)真菌tRNA剪接酶Trl1(tRNA连接酶)的结构、作用机制及其特异性 和Tpt1(tRNA2‘-磷酸转移酶)--作为正常细胞必需的RNA修复系统的范例 生理学，并作为抗真菌药物发现的有希望的目标。我们将确定Trl1和Trl1的结构 来自人类真菌病原体烟曲霉和白色念珠菌的Tpt1与 底物、辅因子和反应中间体。 (3)真核细胞tRNA反密码子核酸酶“核毒素”的作用机制和独特的靶标特异性 金合欢毒素(金合欢毒素；PAT)，是物种自我-非自我区别的基础。我们将确定PAT in的结构 与其底物tRNAGln(UUG)反密码子环形成的复合体。我们将通过以下方式说明保护性免疫的基础 通过解决Pat·ImmPaT杂二聚体的结构，获得了抗ImmPaT的毕赤酵母抗毒素。 (4)RNA聚合酶II(Pol2)CTD编码。Pol2 CTD，由一致的串联七肽组成 序列Y1S2P3T4S5P6S7对活性至关重要，因为它招募了调节转录的蛋白质， 改变染色质结构，催化或调节mRNA的封顶、剪接和多聚腺苷化。通过 通过基因操作裂解酵母CTD，并测量对细胞生长和基因表达的影响，我们：(I) 推导出代码的每一字母的结构-活性关系；以及(Ii)定义字母组合，该组合 包括由细胞因子“阅读”并支配特定表达程序的“词”。我们专注于 CTD和转录因子Pho7在裂解酵母磷稳态中的作用 从而在充满磷的细胞中抑制获得磷的基因(以一种依赖于 CTD磷酸化状态)，并在磷酸盐饥饿时被激活(这一事件取决于Pho7)。

PROJECT SUMMARY: This MIRA proposal consolidates and extends diverse lines of inquiry into fundamental DNA and RNA transactions that were heretofore supported by four longstanding NIGMS grants. The goal of this research is: (i) to understand the mechanisms and structures of enzymes that perform nucleic acid synthesis, modification, and repair; and (ii) to elucidate factors that regulate these events. The project integrates diverse experimental approaches (microbiology, biochemistry, structural biology, genetics) and applies them to model systems ranging from viruses to bacteria to fungi. The principal themes are: (1) The chemical mechanism and structural basis for end recognition by polynucleotide ligases and mRNA capping enzymes that catalyze nucleotidyl transfer to 5' phosphorylated ends via a covalent enzyme-(lysyl- Nζ)–NMP intermediate. We will solve structures of exemplary ATP-dependent DNA ligases and capping enzymes as their step 1 Michaelis complexes with NTP and metal cofactors. We will clarify the specificity of the NHEJ ligase LigD for a 3'-monoribonucleotide nick and of capping enzyme for ppRNA. We will employ time- lapse crystallography to probe the role of metals in phosphodiester synthesis by ligases. (2) The structure, mechanism, and distinctive specificities of fungal tRNA splicing enzymes Trl1 (tRNA ligase) and Tpt1 (tRNA 2'-phosphotransferase) – as paradigms of an RNA repair system essential for normal cell physiology and as promising targets for anti-fungal drug discovery. We will determine structures of Trl1 and Tpt1 from the human fungal pathogens Aspergillus fumigatus and Candida albicans in complexes with substrates, cofactors, and reaction intermediates. (3) The mechanism and distinctive target specificity of a eukaryal tRNA anticodon nuclease “ribotoxin” (Pichia acaciae toxin; PaT) that underlies species self-nonself discrimination. We will determine the structure of PaT in complex with its substrate anticodon loop of tRNAGln(UUG). We will illuminate the basis for protective immunity by the Pichia acaciae antitoxin ImmPaT by solving the structure of a PaT·ImmPaT heterodimer. (4) The RNA polymerase II (Pol2) CTD code. The Pol2 CTD, consisting of tandem heptapeptides of consensus sequence Y1S2P3T4S5P6S7, is essential for viability because it recruits proteins that regulate transcription, modify chromatin structure, and catalyze or regulate mRNA capping, splicing, and polyadenylation. By genetically manipulating the fission yeast CTD, and gauging effects on cell growth and gene expression, we: (i) educed structure-activity relations for each “letter” of the code; and (ii) defined combinations of letters that comprise “words” that are “read” by cellular factors, and which govern specific expression programs. We focus here on the roles of CTD and transcription factor Pho7 in fission yeast phosphate homeostasis, a mechanism whereby phosphate-acquisition genes are repressed in phosphate-replete cells (in a manner dependent on CTD phospho-status), and activated in response to phosphate starvation (an event dependent on Pho7).