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节由RNA聚合酶II亚基1的C末端结构域（CTD）的修饰暂时介导。CTD的组合磷酸化模式，YSPTSPS七肽的52个共有重复序列，协调转录周期中的RNA生物合成。由于缺乏合适的方法对CTD磷酸化进行位点特异性和定量定位，解决CTD调节作用的进展受到阻碍，这是一个将结构与功能直接联系在一起的问题。该提案通过开发紫外光解离（UVPD）质谱来解决这一挑战，以绘制CTD的磷酸化模式和占用率，并结合无标记定量。提出的三个目标包括：（1）使用193 nm UVPD分析CTD的组合磷酸化模式。将分离RNA pol II的~26 kDa CTD肽，用蛋白酶K消化，并在负模式下通过nanoLC-UVPD-MS分析所得肽。将表征磷酸化位点和结合位点。(2)整体CTD磷酰基占有率和暴露于细胞应激源的定量分析。CTD编码的变化作为特定细胞应激源的函数，包括热休克、暴露于盐、二硫苏糖醇、脂多糖（LPS）、山梨糖醇或过氧化氢，将通过无标记定量来阐明。(3)对照和具有功能缺陷的Ssu 72磷酸酶的酵母菌株之间的全局CTD磷酰基占据的定量分析。Ssu 72显示对CTD七肽重复序列的Ser 2和Ser 5的特异性磷酸酶活性，这两个位点在转录的时间调节中起关键作用。将收获来自Ssu 72缺陷型和对照酵母菌株的RNA pol II，用胰蛋白酶消化，并处理以分离CTD肽。将通过无标记定量评价所得磷酸化肽，其在每种细胞状态下的占有率和丰度不同。 创新UVPD技术的应用将为CTD磷酸化在转录过程中的作用提供重要的见解。

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.