The goal is to improve the performance DNA-affinity chromatography of transcription factors and to increase understanding of genetic regulation. To accomplish this we will: 1. Develop protocols we call """"""""rational trapping"""""""" to allow oligonucleotide trapping to be easily optimized for a particular protein. Previously, we had developed a method called """"""""oligonucleotide trapping"""""""" in which a probe DNA is mixed with cell extract at very low concentrations and with various competitors which lessen nonspecific binding. The protein-DNA complex is then rapidly isolated by chromatography and the transcription factor elutes in a high state of purity, typically homogeneous. The current method takes a long time to refine. We will use electrophoretic mobility shift assays to develop a rigorous, rational way to arrive at the correct conditions for successful purification. 2. The application of rational trapping to three very different transcription factors will show if it functions well for diverse proteins. The method will then be applied to three other transcription factors to show that it works well for diverse proteins from diverse organsims. 3. A new method, called promoter trapping, will be developed. The c-jun promoter is only about 200 bp. We will extend this trapping concept to purify the entire transcription pre-initiation complex (PIC) and characterize its component proteins by a proteomics approach using trypsin digestion and the mass spectrometers. This technique will allow identification of all components of the PIC, including ones which are currently unknown. The method, along with rational trapping, will then be used to purify three of the transcription factors bound by this promoter to investigate transcriptional regulation by cell signaling/phosphorylation in HeLa cells in response to serum mitogens. One of these transcription factors is currently unknown and will be characterized here for the first time.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM043609-19
Application #
7254944
Study Section
Special Emphasis Panel (ZRG1-BECM (01))
Program Officer
Edmonds, Charles G
Project Start
1989-08-01
Project End
2009-06-30
Budget Start
2007-07-01
Budget End
2008-06-30
Support Year
19
Fiscal Year
2007
Total Cost
$223,389
Indirect Cost
Name
University of Texas Health Science Center San Antonio
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
800189185
City
San Antonio
State
TX
Country
United States
Zip Code
78249
Jia, Yinshan; Nagore, Linda; Jarrett, Harry (2015) Southwestern Blotting Assay. Methods Mol Biol 1334:85-99
Jia, Yinshan; Jarrett, Harry W (2015) Method for trapping affinity chromatography of transcription factors using aldehyde-hydrazide coupling to agarose. Anal Biochem 482:1-6
Nagore, Linda I; Jarrett, Harry W (2015) T(3): targeted proteomics of DNA-binding proteins. Anal Biochem 474:8-15
Jia, Yinshan; Larionov, Oleg; Jarrett, Harry W (2014) Coupling of deoxyribonucleic acid to solid supports using 3' terminal ribose incorporation. J Chromatogr A 1339:73-9
Nagore, L I; Nadeau, R J; Guo, Q et al. (2013) Purification and characterization of transcription factors. Mass Spectrom Rev 32:386-98
Hoffmann, Christoph; Zimmermann, Anika; Hinney, Anke et al. (2013) A novel SP1/SP3 dependent intronic enhancer governing transcription of the UCP3 gene in brown adipocytes. PLoS One 8:e83426
Jarrett, Harry W (2012) Proteomic methodologies to study transcription factor function. Methods Mol Biol 786:315-34
Zhou, Yanwen; Jia, Yinshan; Jarrett, Harry W (2012) Asymmetric polymerase chain reaction provides alternatives for preparation of (GT)ýýý-tailed duplex DNA promoter for promoter trapping. Anal Biochem 427:133-8
Jiang, Daifeng; Mummidi, Srinivas; Ahuja, Sunil K et al. (2011) CCR5 promoter haplotype transcription complex characterization. J Health Care Poor Underserved 22:73-90
Jiang, Daifeng; Jia, Yinshan; Jarrett, Harry W (2011) Transcription factor proteomics: identification by a novel gel mobility shift-three-dimensional electrophoresis method coupled with southwestern blot and high-performance liquid chromatography-electrospray-mass spectrometry analysis. J Chromatogr A 1218:7003-15

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