Mechanisms of DNA and RNA Transactions

项目摘要：为MIRA更新提出的研究目标是：(I)了解 执行核酸合成、修饰和修复的酶的机制和结构；以及(Ii) 阐明调节这些事件的因素。该项目整合了多种实验方法。 (微生物学、生物化学、结构生物学、遗传学)，并将它们应用于从 病毒到细菌再到真菌。主要的主题是： (1)真菌tRNA剪接酶Trl1(TRNA)的结构、作用机制和特异性 连接酶)和Tpt1(tRNA2‘-磷酸转移酶)--作为正常必需的RNA修复系统的范例 细胞生理学，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 Tpt1与核酸、核苷酸底物和辅因子的络合物中，并努力捕获 反应路径上中间体和过渡态的结构快照。 (2)依赖于ATP的5‘-PO4/3’-OH RNA连接酶T4识别和链连接的结构基础 Rnl1。Rnl1是trna修复酶，t4噬菌体用它来逃避trna损伤宿主的反应。 病毒感染。 (3)依赖GTP的3‘-PO4/5’-OH RNA的独特催化机制、末端特异性及其调控 连接酶RtcB。RtcB家族连接酶存在于所有的系统发育域中。它们是多种RNA的代理人。 交易，包括tRNA剪接(在后生动物和古生菌中)，RNA修复(在细菌中)，非剪接体 (在后生动物未折叠蛋白反应中)的mRNA剪接，以及人类嵌合RNA的形成 可以逆转座进入人类基因组的细胞。 (4)串联转录干扰是裂解酵母磷稳态的控制因素。这个 3个S.pombe pho调节子基因在充满磷的细胞中通过转录在5‘端顺式转录中被抑制。 干扰pho基因启动子的侧翼InncRNA。LncRNA介导的干扰 通过对Pho1的抑制，我们可以灵敏地读出基因对3‘- 处理/终止和用于发现Pol2这一步骤的代理人和调节器的强大工具 转录周期。这些影响包括：(I)Pol2 CTD代码；(Ii)3‘- 处理/终止机制；以及(Iii)由细胞内的肌醇焦磷酸1，5-IP8控制代谢物 信号分子。我们建议研究裂解酵母Asp1激酶/焦磷酸酶 决定IP8的动态以及连接IP8与基因表达的细胞蛋白质和途径。

PROJECT SUMMARY: The goals of the research proposed for the MIRA renewal are: (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 structures, mechanisms, 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 in complexes with nucleic acid and nucleotide substrates and cofactors, and endeavor to capture structural snapshots of intermediates and transition-states along the reaction pathways. (2) The structural basis for RNA recognition and strand joining by ATP-dependent 5'-PO4/3'-OH RNA ligase T4 Rnl1. Rnl1 is a tRNA repair enzyme that the T4 bacteriophage uses to evade a tRNA-damaging host response to virus infection. (3) The unique catalytic mechanism, end-specificity, and regulation of GTP-dependent 3'-PO4/5'-OH RNA ligase RtcB. The RtcB-family ligases are found in all phylogenetic domains. They are agents of diverse RNA transactions, including tRNA splicing (in metazoa and archaea), RNA repair (in bacteria), nonspliceosomal mRNA splicing (in the metazoan unfolded protein response), and the formation of chimeric RNAs in human cells that can undergo retrotransposition into the human genome. (4) Tandem transcriptional interference as a controlling factor in fission yeast phosphate homeostasis. The three S. pombe PHO regulon genes are repressed in phosphate-replete cells by transcription in cis of 5’- flanking lncRNAs that interferes with the PHO mRNA promoters. The lncRNA-mediated interference that underlies the repression of pho1 has afforded us a sensitive read-out of genetic influences on 3'- processing/termination and a powerful tool for discovery of agents and regulators of this step of the Pol2 transcription cycle. These influences include: (i) the Pol2 CTD code; (ii) numerous components of the 3'- processing/termination machinery; and (iii) metabolite control by inositol pyrophosphate 1,5-IP8, an intracellular signaling molecule. We propose to investigate the fission yeast Asp1 kinase/pyrophosphatase enzyme that determines IP8 dynamics and the cellular proteins and pathways that connect IP8 to gene expression.