We have identified a protein kinase activity which adds multiple phosphate groups to the carboxyl-terminal repeat domain (CTD) of the largest subunit of eukaryotic RNA polymerase II. We plan to purify and characterize this kinase from both yeast and Drosophila and to clone and sequence the gene encoding it. We will then use this information and the genetic manipulations available to yeast and Drosophila to help elucidate the physiological roles of the kinase, focusing in particular on how CTD phosphorylation modulates the properties of RNA polymerase II. Knowledge gained from these investigations, by revealing mechanisms regulating functional properties of the transcription apparatus, will contribute to our understanding of gene expression in all eukaryotes.
SPECIFIC AIMS : 1. Complete the purification of the kinase. 2. Prepare antibodies against the purified enzyme. 3. Clone the kinase gene from yeast and Drosophila using the antibodies and expression vector Libraries. 4. A. Sequence the yeast gene; compare the deducted amino acid sequence to known kinases and oncogenes. B. Disrupt the yeast gene, reintroduce it into the genome, and investigate the null phenotype (anticipate null-lethal). C. Construct conditional mutants; use them to investigate consequences of inactivating the kinase (especially effects on RNA polymerase II). 5. Map the Drosophila gene by in situ hybridization; attempt to correlate it with a known genetic locus. 6. Generate mutants in the Drosophila locus (or use pre-existing ones); investigate their phenotypes and effects on RNA polymerase II.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM040505-03
Application #
3298099
Study Section
Biochemistry Study Section (BIO)
Project Start
1988-07-01
Project End
1991-11-30
Budget Start
1990-07-01
Budget End
1991-11-30
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Duke University
Department
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
Bartkowiak, Bartlomiej; Greenleaf, Arno L (2015) Expression, purification, and identification of associated proteins of the full-length hCDK12/CyclinK complex. J Biol Chem 290:1786-95
Bartkowiak, Bartlomiej; Yan, Christopher; Greenleaf, Arno L (2015) Engineering an analog-sensitive CDK12 cell line using CRISPR/Cas. Biochim Biophys Acta 1849:1179-87
Liu, Jiangxin; Fan, Shilong; Lee, Chul-Jin et al. (2013) Specific interaction of the transcription elongation regulator TCERG1 with RNA polymerase II requires simultaneous phosphorylation at Ser2, Ser5, and Ser7 within the carboxyl-terminal domain repeat. J Biol Chem 288:10890-901
Winsor, Tiffany Sabin; Bartkowiak, Bartlomiej; Bennett, Craig B et al. (2013) A DNA damage response system associated with the phosphoCTD of elongating RNA polymerase II. PLoS One 8:e60909
Möller, André; Xie, Sheila Q; Hosp, Fabian et al. (2012) Proteomic analysis of mitotic RNA polymerase II reveals novel interactors and association with proteins dysfunctional in disease. Mol Cell Proteomics 11:M111.011767
Bartkowiak, Bartlomiej; Greenleaf, Arno L (2011) Phosphorylation of RNAPII: To P-TEFb or not to P-TEFb? Transcription 2:115-119
MacKellar, April L; Greenleaf, Arno L (2011) Cotranscriptional association of mRNA export factor Yra1 with C-terminal domain of RNA polymerase II. J Biol Chem 286:36385-95
Werner-Allen, Jon W; Lee, Chul-Jin; Liu, Pengda et al. (2011) cis-Proline-mediated Ser(P)5 dephosphorylation by the RNA polymerase II C-terminal domain phosphatase Ssu72. J Biol Chem 286:5717-26
Bartkowiak, Bartlomiej; Mackellar, April L; Greenleaf, Arno L (2011) Updating the CTD Story: From Tail to Epic. Genet Res Int 2011:623718
Liu, Pengda; Kenney, John M; Stiller, John W et al. (2010) Genetic organization, length conservation, and evolution of RNA polymerase II carboxyl-terminal domain. Mol Biol Evol 27:2628-41

Showing the most recent 10 out of 24 publications