mRNA Capping Enzyme

描述（由申请人提供）： 项目摘要。该项目的长期目标是了解RNA聚合酶II（RNApII）的转录如何与RNA加工和终止相结合。该项目的早期资助期产生了一个模型，其中RNApII亚基Rpb 1的C-末端结构域（CTD）在转录周期的不同阶段显示特征磷酸化模式，以促进适当因子的结合，用于共转录RNA加工。这些研究是在酿酒酵母的模型系统中进行的，但这个过程在进化过程中是高度保守的。该项目产生的基础知识提供了重要的洞察CTD磷酸化循环如何影响医学上重要的过程，如刺激HIV转录的病毒达特蛋白和“暂停”RNApII在胚胎干细胞发育调控基因。为了更好地了解介导CTD磷酸化变化的酶（激酶、磷酸酶等），以及识别这些模式的蛋白质。 在下一个供资期内，提出了四个具体目标。第一个是继续研究，以了解两种已知的终止途径的机制，以及RNApII如何在它们之间进行选择：用于小的非编码转录本和许多“隐藏”转录本的早期终止途径，或用于mRNA的后来的多聚腺苷酸化偶联终止途径。第二个相关的目的是研究许多RNApII启动子的不同转录产生的短的不稳定转录本的生物起源，重点是它们可以告诉我们关于起始、早期延伸和终止的信息。第三个目标将使用一种新的高分辨率技术来定位新生转录本的3'端，以进一步探测CTD磷酸化在RNA延伸、暂停和终止中的作用。第四个具体目标建立在令人惊讶的发现之上，即Rpb 1 CTD可以转移到不同的RNApII亚基上并且仍然起作用。这一发现形成了一组实验的基础，这些实验询问不同的CTD修饰和功能是否需要在相同的CTD上以“顺式”发生，或者可以在两个CTD之间分裂，以及这些磷酸化是否可以通过将它们的靶标直接连接到RNApII来绕过。

DESCRIPTION (provided by applicant): Project Summary. The long-term goal of this project is to understand how transcription by RNA polymerase II (RNApII) is coupled to RNA processing and termination. Earlier funding periods of this project produced a model in which the C-terminal domain (CTD) of the RNApII subunit Rpb1 displays characteristic phosphorylation patterns at different stages of the transcription cycle to promote binding of the appropriate factors for co-transcriptional RNA processing. These studies are done in the model system Saccharomyces cerevisiae, but this process is highly conserved over evolution. The fundamental knowledge generated by this project provides significant insight into how the CTD phosphorylation cycle affects medically important processes such as the stimulation of HIV transcription by the viral Tat protein and the "pausing" of RNApII at developmentally regulated genes in embryonic stem cells. Continuation of this project is necessary to better understand both the enzymes that mediate the changes in CTD phosphorylation (kinases, phosphatases, etc.) as well as the proteins that recognize these patterns. In the next funding period, four specific aims are proposed. The first is a continuation of studies to understand the mechanisms of the two known termination pathways and how RNApII chooses between them: an early termination pathway used for small noncoding transcripts and many "cryptic" transcripts or the later polyadenylation-coupled termination pathway used for mRNAs. A second related aim is to study the biogenesis of short unstable transcripts produced by divergent transcription of many RNApII promoters, with a focus on what they can tell us about initiation, early elongation, and termination. The third aim will use a new, high- resolution technique for mapping 3' ends of nascent transcripts to further probe the role of CTD phosphorylation in RNA elongation, pausing, and termination. The fourth specific aim builds upon the surprising discovery that the Rpb1 CTD can be transferred onto a different RNApII subunit and still function. This finding forms the basis for a set of experiments asking whether the different CTD modifications and functions need to occur in "cis" on the same CTD or can be split between two CTDs, and whether these phosphorylations can be bypassed by tethering their targets directly to RNApII.