Biochemical Analysis of PHO5 Gene Regulation

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This research will investigate the function of the core histones in the regulation of RNA polymerase II transcription. Many, if not most, transcription regulatory mechanisms may be fully manifest only in a chromatin context. Core histones in the form of nucleosomes repress transcription. Recent genetics studies indicate that core histones are directly involved in RNA polymerase II transcription regulation. The mechanism of transcription regulation on a chromatin template will be investigated using Pho4p transcription factor activation of the yeast PHOS promoter as a model. Results from these studies will provide a more precise understanding of the role of the core histones in the mechanisms that determine whether a gene is active or repressed. Depletion of core histones in vivo causes the derepression of transcription of the yeast PHO5, GAL 1, and CYC 1 genes. Previous studies of PHO5 gene regulation have provided important clues to the mechanism of transcription regulation on chromatin. However, these studies have all been carried out in vivo. A more complete understanding o f the mechanism requires biochemical analysis. This laboratory is the first to have purified yeast core histones in large enough quantities for biochemical investigation of their role transcription regulation. These histones have been used to reconstitute nucleosomal templates, restoring repression of basal transcription to the PHO5 promoter. Thus, this laboratory is in a unique position to undertake these analyses. This research will: (1) Reconstitute transcription activation from the PHO5 promoter. In vitro transcription will be reconstituted from recombinant and purified yeast basal transcription factors and purified Pho4p and Pho2p, and these systems will be used to reconstitute basal and activated PHO5 transcription. (2) Determine the degree of PHO5 basal transcription repression by reconstituted nucleosomes. Nucleosomes will be reconstituted on the PHOS promoter using purified components. The levels of basal transcription between naked DNA and chromatin templates will be compared, and the relation between TATA-box and RNA start site accessibility and transcription activity on chromatin templates will be determined. (3) Investigate the ability of Pho4p to counteract nucleosome repression of the PHO5 promoter. Nucleosomes will be reconstituted onto the PHO5 promoter, and the factor requirement will be determined for Pho4p to counteract nucleosome repression. The level of PHO5 activation will be compared on naked and chromatin templates. * * * 9506255 Richter This research addresses the relationship between the structure, function and regulation of the ATP synthase enzyme. This allosteric enzyme reversibly utilizes the energy of a transmembrane proton gradient to synthesize ATP. The long term goal of the research is to contribute toward an understanding of the mechanism of the energy transduction process and to understand how this enzyme is regulated under physiological conditions in plants and animals. Prior NSF funding has led to: 1) Cloning and over-expression of all five subunits of the catalytic F1 portion of the chloroplast ATP synthase (CF1); 2) Recovery of the subunits from bacterial inclusion bodies and refolding them into their native, active forms; and 3) Reconstituting them into a functional F1 complex. Further studies will utilize this system to genetically engineer CF1 subunits for analyzing regions of functional importance, and to examine functional dynamics of subunits during the catalytic process using biochemical and biophysical techniques. This proposal describes a collaborative multidiciplinary approach focusing on the structure and function of the three smaller CF1 subunits, )and (. Genetic engineering experiments will involve: 1) Site-directed mutagenesis studies to probe the functional and structural importance of different regions of the polypeptide chains via replacement or deletion of specific amino acids or sequences of amino acids; 2) Studies of subunit dynamics through attachment of intrinsic (tryptophans) and extrinsic (via cysteine residues) fluorescent probes to specific sites on the ( ( and ( subunits. Labeled subunits will be reconstituted with CF1 deficient in these subunits for fluorescence studies aimed at (a) structural mapping of subunits within the CF1 complex using fluorescence resonance energy transfer measurements and (b) monitoring subunit dynamics during catalytic turnover by the enzyme by monitoring the time- dependent anisotropy of intrinsic and extrinsic fluorescent probes; 3) Chemical crosslinking studies involving attachment of bifunctional chemical crosslinking agents at various engineered sites within the ( and ( subunits. Mutant subunits will be reconstituted with the other CF1 subunits and with the membrane-bound proton channel portion (CF0) of the enzyme for chemical crosslinking studies aimed at identifying the sites of interaction between CF1 and CF0 subunits. %%% This research addresses the relationship between the chemical structure and biological function of the ATP synthase enzyme of plant chloroplast membranes. This enzyme is responsible for the conversion of the energy from sunlight into the chemical storage form. ATP (adenosine triphosphate). ATP in turn supplies the energy for the conversion of carbon dioxide into sugar in plants. Analogous enzymes supply more than 90% of the ATP required for energy-dependent metabolic processes in animals and bacteria. The long term goal of the research described in this proposal is to identify, at the molecular level, how the ATP synthase enzymes capture energy and convert it into ATP. We intend to approach this goal by genetically engineering the ATP synthase so as to probe the importance of specific sites within the enzyme for the energy conversion (catalytic) process. Some of the genetically engineered sites will provide sites for attachment of chemical probes which will be used to identify specific events involving changes in the structure of the enzyme which occur during the catalytic process. Attachment of other chemical probes to genetically engineered sites will allow us to cross-link (tether) pieces of the enzyme together so that we can identify specific sites of protein-protein interaction which occur within the enzyme and which are important for catalysis. A thorough understanding of this very efficient natural energy conserving process will potentially lead to the design and implementation of vastly improved energy storage and utilization processes or human use.