Deciphering the RNA Polymerase II CTD Code

 描述（由申请人提供）：RNA聚合酶II（Pol 2）最大亚基的羧基末端结构域（CTD）由共有序列Y1 S2 P3 T4 S5 P6 S7的串联重复七肽组成。CTD对细胞活力至关重要，因为它募集调节转录、修饰染色质结构以及催化或调节mRNA加帽、剪接和多腺苷酸化的蛋白质。固有的可塑性CTD结构通过七肽丝氨酸（S2、S5、S7）、苏氨酸（T4）和酪氨酸（Y1）残基的动态磷酸化和去磷酸化来调节。CTD的磷酸化状态提供了关于转录机制的信息线索-“CTD代码”-其被CTD受体蛋白“读取”。我们在这个项目中的目标是了解CTD信息是如何被写入，组织和转导到细胞效应器，以及CTD代码如何控制基因表达。我们正在通过基因操作裂变酵母Pol 2 CTD的组成和结构来破译密码。这种方法告诉我们：（i）Ser 2、Thr 4和Ser 7磷酸化位点对于裂殖酵母的生存力不是必需的;（ii）Phe代替Tyr 1发挥作用;（iii）Ser 5是唯一严格必需的磷酸化标记，其标记需要Pro 6。我们已经证明Ser 5-PO 4标记的主要功能是将mRNA加帽装置募集到新生Pol 2转录物中。现在突出的挑战是了解CTD编码字母如何组装成“单词”（即，词汇表）。这个项目旨在通过两种互补的方法来定义单个编码字母的生理学，以及CTD词汇的雏形。为了测量代码的输出，我们将对我们收集的裂变酵母CTD突变体进行转录组分析。为了获得依赖于特定CTD线索的基因表达步骤的新见解，我们将分析Pol 2 CTD突变体的突变协同作用（例如，合成致死相互作用）及其等位基因特异性。初步结果表明，fssion酵母磷酸盐和铁稳态调节，这是由特定的DNA结合转录因子控制，强烈影响个别CTD标记的突变。我们将解剖生化和结构的Pho 7和Fep 1转录因子，控制磷酸盐和铁调节子，并探测其功能/物理相互作用与CTD。最后，我们将阐明的结构，机制和特异性的基本裂变酵母CTD磷酸酶Fcp 1。

 DESCRIPTION (provided by applicant): The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol2) consists of tandemly repeated heptapeptides of consensus sequence Y1S2P3T4S5P6S7. The CTD is essential for cell viability because it recruits proteins that regulate transcription, modify chromatin structure, and catalyze or regulate mRNA capping, splicing, and polyadenylation. The inherently plastic CTD structure is modulated by dynamic phosphorylation and dephosphorylation of the heptad serine (S2, S5, S7), threonine (T4), and tyrosine (Y1) residues. The phospho-status of the CTD provides informational cues about the transcription machinery - a "CTD code" - that is "read" by CTD receptor proteins. Our goals in this project are to understand how CTD information is inscribed, organized, and transduced to cellular effectors, and how the CTD code governs gene expression. We are deciphering the code by genetically manipulating the composition and structure of the fission yeast Pol2 CTD. This approach has taught us that: (i) the Ser2, Thr4, and Ser7 phospho-sites are not essential for fission yeast viability; (ii) Phe is functional in lieu of Tyr1; and (iii) Ser5 is the only strctly essential phosphorylation mark, inscription of which requires Pro6. We've shown that the chief function of the Ser5-PO4 mark is to recruit the mRNA capping apparatus to nascent Pol2 transcripts. The outstanding challenge now is to understand how CTD coding letters are assembled into "words" (i.e., a vocabulary). This project aims to define the physiology of individual coding letters, and the rudiments of a CTD vocabulary, via two complementary approaches. To gauge the output of the code, we will perform transcriptome profiling of our collection of fission yeast CTD mutants. To gain new insights to steps in gene expression that rely on particular CTD cues, we will analyze mutational synergies of Pol2 CTD mutants (e.g., synthetic lethal interactions) and their allele-specificities. Initial results highlight that the fssion yeast phosphate and iron homeostasis regulons, which are controlled by specific DNA-binding transcription factors, are strongly influenced by mutation of individual CTD marks. We will dissect biochemically and structurally the Pho7 and Fep1 transcription factors that control the phosphate and iron regulons, and probe their functional/physical interactions with the CTD. Finally, we will illuminate the structure, mechanism, and specificity of the essential fission yeas CTD phosphatase Fcp1.