The CTD of RNA polymerase II is an unusual repeat domain that extends, tentacle-like, out from the globular body of the polymerase and acts as a scaffold to which nuclear factors bind; these associations physically link the factors to the transcription machinery. The CTD of elongating RNAP II is highly phosphorylated, as a result of the action of several CTD kinases, and it appears that different patterns of CTD phosphorylation effect recruitment of distinct factors to the transcribing polymerase. However, current understanding of the process of CTD phosphorylation is quite fragmentary. Moreover, while the functions of dozens of nuclear proteins are undoubtedly influenced by phosphoCTD binding, only a few phosphoCTD-associating proteins (PCAPs) have been characterized. Our overall goal is to generate extensive new information on CTD kinases and PCAPs that will bring about enhanced understanding of CTD phosphorylation and phosphoCTD function; we believe that this in turn will engender novel insights into a large number of fundamental nuclear functions and events. To achieve this goal we plan the following Specific Aims: ? ? 1. Determine the modes and consequences of CTD phosphorylation by CTD Kinase I. We will describe with biochemical rigor the disposition of phosphates on the CTD in vitro and in vivo. ? ? 2. Characterize binding domains and properties of novel PCAPs. We will identify phosphoCTD-interacting domains (PCIDs), then determine their phospho-epitope binding specifity and 3D structure. ? ? 3. Investigate and describe PCAP binding and functions in vivo. We will characterize association of PCAPs with target genes in vivo and assess how PCTD binding contributes to PCAP functions. Since the CTD and its transactions are highly conserved from yeast to man, this research is directly relevant to human biology and disease; it will contribute ultimately to betterment of human health. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM040505-16
Application #
6979834
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Tompkins, Laurie
Project Start
1988-07-01
Project End
2009-08-31
Budget Start
2005-09-01
Budget End
2006-08-31
Support Year
16
Fiscal Year
2005
Total Cost
$484,382
Indirect Cost
Name
Duke University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
044387793
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

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