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.

Public Health Relevance

The transcription of DNA and gene expression required for human life are regulated in large part by information coded in the C terminal domain of RNA polymerase II (subunit 1). The proposed research develops ultraviolet photodissociation to map the phosphorylation pattern of the C terminal domain and thereby decipher its transcription code.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21EB018391-01
Application #
8676088
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Liu, Christina
Project Start
2014-06-01
Project End
2016-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Texas Austin
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Austin
State
TX
Country
United States
Zip Code
78712
Mayfield, Joshua E; Robinson, Michelle R; Cotham, Victoria C et al. (2017) Mapping the Phosphorylation Pattern of Drosophila melanogaster RNA Polymerase II Carboxyl-Terminal Domain Using Ultraviolet Photodissociation Mass Spectrometry. ACS Chem Biol 12:153-162
Portz, Bede; Lu, Feiyue; Gibbs, Eric B et al. (2017) Structural heterogeneity in the intrinsically disordered RNA polymerase II C-terminal domain. Nat Commun 8:15231
Cotham, Victoria C; McGee, William M; Brodbelt, Jennifer S (2016) Modulation of Phosphopeptide Fragmentation via Dual Spray Ion/Ion Reactions Using a Sulfonate-Incorporating Reagent. Anal Chem 88:8158-65
Robinson, Michelle R; Taliaferro, Juliana M; Dalby, Kevin N et al. (2016) 193 nm Ultraviolet Photodissociation Mass Spectrometry for Phosphopeptide Characterization in the Positive and Negative Ion Modes. J Proteome Res 15:2739-48
Brodbelt, Jennifer S (2016) Ion Activation Methods for Peptides and Proteins. Anal Chem 88:30-51