Mapping the C Terminal Domain of RNA Polymerase II by UVPD Mass Spectrometry

描述（由申请人提供）：基因表达的转录和调控是通过RNA聚合酶II亚基1的C末端结构域（CTD）的修饰暂时介导的。CTD的组合磷酸化模式是52个一致重复的YSPTSPS七肽，在转录周期中协调RNA的生物发生。由于缺乏CTD磷酸化位点特异性和定量定位的合适方法，解决CTD调控作用的进展受到阻碍，这是一个直接将结构与功能联系起来的问题。本提案通过发展紫外光解（UVPD）质谱法来解决这一挑战，以结合无标记定量来绘制CTD的磷酸化模式和占用。提出的三个目标包括：(1)利用193nm UVPD分析CTD的组合磷酸化模式。将RNA pol II的~26 kDa的CTD肽分离出来，用蛋白酶K消化，用nanoLC-UVPD-MS在阴性模式下分析所得肽。磷酸化位点和占位都将被表征。(2)全球CTD磷酸化占用和细胞应激暴露的定量分析。CTD编码的变化作为特定细胞应激源的功能，包括热休克、暴露于盐、二硫苏糖醇、脂多糖（LPS）、山梨醇或过氧化氢，将通过无标记定量来阐明。(3) Ssu72磷酸酶功能缺陷酵母株与对照株间CTD全域磷酸化占用率的定量分析。Ssu72对CTD七肽重复序列的Ser2和Ser5表现出特异性的磷酸酶活性，这两个位点在转录的时间调控中起着关键作用。从ssu72缺陷和对照酵母菌株中收集RNA pol II，用胰蛋白酶消化，并处理以分离CTD肽。由此产生的磷酸化肽，其在每个细胞状态下的占用和丰度不同，将通过无标记定量进行评估。这种创新的UVPD技术的应用将为CTD磷酸化在转录过程中的作用提供关键的见解。

DESCRIPTION (provided by applicant): Transcription and regulation of gene expression are temporally mediated by modification of the C terminal domain (CTD) of subunit 1 of RNA polymerase II. The combinatorial phosphorylation pattern of the CTD, a 52 consensus repeat of a YSPTSPS heptad, coordinates RNA biogenesis in the transcription cycle. Progress in resolving the regulatory roles of the CTD has been impeded by lack of suitable methods for site-specific and quantitative mapping of CTD phosphorylation, an issue that directly ties structure to function. This proposal addresses this challenge by the development of ultraviolet photodissociation (UVPD) mass spectrometry to map the phosphorylation pattern and occupancy of the CTD in conjunction with label-free quantitation. The three proposed aims include: (1) Analysis of the combinatorial phosphorylation pattern of the CTD using 193 nm UVPD. The ~26 kDa CTD peptide of RNA pol II will be isolated, digested by Proteinase K, and the resulting peptides analyzed by nanoLC-UVPD-MS in the negative mode. Both the phosphorylation sites and occupancies will be characterized. (2) Quantitative analysis of global CTD phosphoryl occupancy and exposure to cell stressors. Changes in the CTD code as a function of particular cell stressors, including heat shock, exposure to salt, dithiothreitol, lipopolysaccharides (LPS), sorbitol or hydrogen peroxide, will be elucidated by label-free quantitation. (3) Quantitative analysis of global CTD phosphoryl occupancy between a control and a yeast strain with a functionally deficient Ssu72 phosphatase. Ssu72 displays specific phosphatase activity toward Ser2 and Ser5 of the CTD heptad repeat, two sites that play a critical role in the temporal regulation of transcription. RNA pol II from Ssu72-deficient and control yeast strains will be harvested, digested with trypsin, and processed to isolate the CTD peptides. The resulting phosphorylated peptides, which will differ in occupancy and abundance for each cell state, will be evaluated by label-free quantitation. This application of innovative UVPD technology will provide critical insight into the role of CTD phosphorylation in transcriptional processing.