title: Mapping the Phosphorylation Pattern of Drosophila melanogaster RNA Polymerase II Carboxyl-Terminal Domain Using Ultraviolet Photodissociation Mass Spectrometry.
abstract: Phosphorylation of the C-terminal domain of RNA polymerase II (CTD) plays an essential role in eukaryotic transcription by recruiting transcriptional regulatory factors to the active polymerase. However, the scarcity of basic residues and repetitive nature of the CTD sequence impose a huge challenge for site-specific characterization of phosphorylation, hindering our understanding of this crucial biological process. Herein, we apply LC-UVPD-MS methods to analyze post-translational modification along native sequence CTDs. Application of our method to the Drosophila melanogaster CTD reveals the phosphorylation pattern of this model organism for the first time. The divergent nature of fly CTD allows us to derive rules defining how flanking residues affect phosphorylation choice by CTD kinases. Our data support the use of LC-UVPD-MS to decipher the CTD code and determine rules that program its function.
authors.forename: Joshua E
authors.initials: JE
authors.lastname: Mayfield
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Robinson
authors.forename: Victoria C
authors.initials: VC
authors.lastname: Cotham
authors.forename: Seema
authors.initials: S
authors.lastname: Irani
authors.forename: Wendy L
authors.initials: WL
authors.lastname: Matthews
authors.forename: Anjana
authors.initials: A
authors.lastname: Ram
authors.affiliation: Department of Biochemistry and Molecular Biology, Penn State University , University Park, Pennsylvania 16802, United States.
authors.forename: David S
authors.initials: DS
authors.lastname: Gilmour
authors.forename: Joe R
authors.initials: JR
authors.lastname: Cannon
authors.forename: Yan Jessie
authors.identifier: 0000-0002-9360-5388
authors.initials: YJ
authors.lastname: Zhang
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Brodbelt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004331:Drosophila melanogaster / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D010766:Phosphorylation; D000072417:Protein Domains; D011499:Protein Processing, Post-Translational; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D019950:Mitogen-Activated Protein Kinase 1; D012319:RNA Polymerase II