Despite a great deal of work, many facets of the mechanism of activated transcription remain completely unclear, and yet this is a central problem that must be solved for a proper understanding of gene regulation. In order to increase our understanding of this fundamental process, we are setting up an efficient system for in vitro transcription using yeast as a model system, specifically the yeast CUP1 gene. We chose this gene because its biological function (the gene encodes a copper-metallothionein) and its mechanism of induction are relatively simple and well-understood at the molecular level: copper ions bind to the DNA-binding domain of a transcriptional activator (Ace1p) which then folds and is able to recognize upstream activating sequences (UASs) in the CUP1 promoter. Ace1p is small (225 residues) with two domains; the other domain is a typical acidic activation domain. It is our aim to reconstitute this system in vitro using purified homologous (yeast) components and using as template either a plasmid containing CUP1, or minichromosomes containing CUP1 isolated from induced and uninduced cells. Such a system should also allow us to evaluate the role of chromatin structure in the regulation of a eukaryotic gene. Toward these ends, we have engineered a yeast strain containing a tagged gene for the large subunit of RNA polymerase II (RPB1). This has allowed us to isolate the yeast RNA polymerase II holoenzyme rapidly and effectively. This is an essential step toward setting up an efficient transcription system. We have purified recombinant Ace1p and we are in the process of preparing other recombinant yeast transcription factors (yTBP, yTFIIB, yTFIIE, etc.). A method for the isolation of yeast minichromosomes in relatively large quantities is also in the final stage of development. In a collaborative venture with Drs. Clore and Gronenborn, we are preparing the DNA-binding domain of Ace1p to solve the structure of its complex with DNA by two-dimensional NMR. This should be a very interesting and unique structure because of the role of copper ions in folding the domain.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Intramural Research (Z01)
Project #
1Z01DK015700-01
Application #
2439078
Study Section
Special Emphasis Panel (LCDB)
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
1996
Total Cost
Indirect Cost
City
State
Country
United States
Zip Code
Sun, Yu; Bak, Beata; Schoenmakers, Nadia et al. (2012) Loss-of-function mutations in IGSF1 cause an X-linked syndrome of central hypothyroidism and testicular enlargement. Nat Genet 44:1375-81
Liu, Y V; Clark, D J; Tchernajenko, V et al. (2003) Role of C-terminal domain phosphorylation in RNA polymerase II transcription through the nucleosome. Biopolymers 68:528-38
Kim, Yeonjung; Clark, David J (2002) SWI/SNF-dependent long-range remodeling of yeast HIS3 chromatin. Proc Natl Acad Sci U S A 99:15381-6
Shen, Chang-Hui; Leblanc, Benoit P; Neal, Carolyn et al. (2002) Targeted histone acetylation at the yeast CUP1 promoter requires the transcriptional activator, the TATA boxes, and the putative histone acetylase encoded by SPT10. Mol Cell Biol 22:6406-16
Oliver, Brian; Parisi, Michael; Clark, David (2002) Gene expression neighborhoods. J Biol 1:4
Shen, C H; Clark, D J (2001) DNA sequence plays a major role in determining nucleosome positions in yeast CUP1 chromatin. J Biol Chem 276:35209-16
Shen, C H; Leblanc, B P; Alfieri, J A et al. (2001) Remodeling of yeast CUP1 chromatin involves activator-dependent repositioning of nucleosomes over the entire gene and flanking sequences. Mol Cell Biol 21:534-47
Leblanc, B P; Benham, C J; Clark, D J (2000) An initiation element in the yeast CUP1 promoter is recognized by RNA polymerase II in the absence of TATA box-binding protein if the DNA is negatively supercoiled. Proc Natl Acad Sci U S A 97:10745-50
Alfieri, J A; Clark, D J (1999) Isolation of minichromosomes from yeast cells. Methods Enzymol 304:35-49