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

描述(申请人提供)：基因表达的转录和调节是通过修改RNA聚合酶II亚单位1的C末端结构域(CTD)来暂时调节的。CTD的组合磷酸化模式，YSPTSPS七肽的52个共识重复，在转录周期中协调RNA的生物发生。由于缺乏合适的方法来定位和定量定位CTD磷酸化，阻碍了解决CTD调控作用的进展，这是一个直接将结构与功能联系在一起的问题。这项建议通过发展紫外光解离(UVPD)质谱学来绘制CTD的磷酸化模式和占有率，并结合无标记定量来解决这一挑战。提出的三个目标包括：(1)利用193 nm UVPD分析CTD的组合磷酸化模式。将RNA polII的~26 kDa CTD多肽分离出来，用蛋白酶K消化，得到的多肽用NanoLC-UVPD-MS进行负性分析。将对磷酸化位置和占有率进行表征。(2)全球CTD磷酸化占有率和细胞应激源暴露的定量分析。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)、山梨醇或过氧化氢，将通过无标记定量来阐明。(3)定量分析对照和Ssu72磷酸酶功能缺陷的酵母菌株之间的整体CTD磷酸化占有率。Ssu72对CTD七肽重复序列的Ser2和Ser5显示出特异性的磷酸酶活性，这两个位置在转录的时间调控中起着关键作用。从Ssu72缺陷酵母菌株和对照酵母菌株中提取RNA polII，用胰酶消化，处理后分离CTD多肽。由此产生的磷酸化多肽将通过无标记定量进行评估，这些多肽在每个细胞状态下的占有率和丰度都不同。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.