Despite a great deal of work, many facets of the mechanism of activated transcription remain unclear, and yet this is a central problem that must be solved for a complete understanding of gene regulation. To address this problem, we are setting up an efficient system for in vitro transcription using yeast as a model system, specifically the yeast CUP1 gene, which encodes a copper-metallothionein. We chose this gene because its biological function is relatively simple (to protect the cell against the toxic effects of copper) and its mechanism of induction is well- understood at the molecular level: copper ions bind to the DNA- binding domain of a transcriptional activator (Ace1p) which then folds and recognizes upstream activating sequences (UASs) in the CUP1 promoter. The C-terminal domain of Ace1p is a typical acidic activation domain. It is our aim to reconstitute the regulated transcription of this gene in vitro using native minichromosomes containing CUP1 isolated from induced and uninduced cells as templates, and purified yeast transcription factors. Such a system should allow us to evaluate the role of chromatin structure in the regulation of a eukaryotic gene. We have developed a method for the isolation of yeast minichromosomes in relatively large quantities. The minichromosomes are intact and relatively pure. Thus, we have isolated a gene as native, intact, presumably transcriptionally competent, chromatin - this represents a significant technical breakthrough. We have mapped the nucleosome positions in isolated minichromosomes from cells induced or not induced with copper ions. In both cases the CUP1 promoter is in an open conformation (apparently free of nucleosomes), and preliminary DNase I footprinting experiments suggest the presence of non-histone proteins at the promoter. We are using Western blot analysis to ascertain the presence of Ace1, RNA polymerase II and TBP in our minichromosome preparations. We are attempting transcription run- off experiments to detect stalled RNA polymerase II complexes to determine whether the chromatin is transcriptionally active. Concurrently, we have purified many of the yeast general transcription factors. We have engineered yeast strains containing genes with six-histidine tags to facilitate purification of RNA polymerase II (holoenzyme and core enzyme), TFIIF and TFIIH. We have purified recombinant Ace1p and yTBP, yTFIIA, yTFIIB and yTFIIE. We are now attempting to reconstitute transcription of CUP1 in minichromosomes in vitro. We are beginning a study of the histone acetylation patterns in the minichromosome. For these experiments, minichromosomes containingHIS3, a gene known to be dependent on Gcn5p (a histone acetyltransferase) for full activity, will be used. We hope to test the current model for the mechanism of transcriptional stimulation by Gcn5 directly using this system.
