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Analysis of Functional Features of CTD Kinases by Irreversible Imbibition Strategy
Author(s) -
Liu Jiayue,
Rodriguez Juan,
Gregorio Josefat,
Tseng Sandra,
Ansari Aseem
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.877.17
Subject(s) - ctd , phosphorylation , rna polymerase ii , kinase , transcription (linguistics) , biology , microbiology and biotechnology , biochemistry , chemistry , promoter , gene , gene expression , oceanography , linguistics , philosophy , geology
The C‐terminal domain (CTD) of the largest subunit of RNA polymerase II, Rpb1, consists of a repeating and highly conserved heptapeptide (YSPTSPS) which serves as a binding scaffold for a variety of protein‐protein interactions during transcription. In the eukaryotic model Saccharomyces cerevisiae, the CTD is composed of 26 heptapeptide repeats in tandem. Five residues within the heptapeptide are phosphorylated ( Y 1 S 2 P 3 T 4 S 5 P 6 S 7 ; underlined), whereas proline residues undergo cis/trans isomerization. The dynamic patterns of CTD modifications, in particular phosphorylation and de‐phosphorylation, regulate most of the steps in the transcription cycle (initiation, elongation and termination) and the co‐transcriptional processing of the nascent RNA. To uncover the functional features of CTD kinases, we have applied a novel strategy in which we irreversibly block CTD kinase activity in vivo. We have targeted three kinases that play critical roles in CTD phosphorylation at different stages of transcription: Kin28, Bur1 and Ctk1. Unlike reversible inhibition approaches, we are able to recapitulate the lethal phenotypes associated with catalytically inactive alleles of Kin28 and Bur1 by using a covalent inhibition approach. Consistently and in contrast to reversible inhibition strategies, we observe a more dramatic depletion of phospo‐CTD marks upon targeted covalent inhibition of each CTD kinase in vivo. Our findings suggest (1) covalent inhibition of CTD kinases will reveal additional functional features of these enzymes in cells, and (2) covalent kinase inhibition could be used as a general approach to study kinase function in vivo.