The mechanisms by which eukaryotic organisms regulate gene expression are important for basic scientific knowledge that is necessary for understanding many complex biological phenomena including human diseases. With regard to the process of transcriptional initiation, a wide variety of experiments have pointed to common molecular mechanisms in eukaryotic organisms ranging from humans to yeasts. This proposal will continue to investigate several basic issues concerning the molecular mechanisms of transcriptional regulation in yeast by combining a wide variety of techniques including recombinant DNA technology, molecular and classical yeast genetics, and protein and nucleic acid biochemistry. First, we will investigate the various functions of the TFIID by obtaining mutants with the following properties: support basal transcription but fail to respond to acidic activator proteins; are specifically defective either for transcription by RNA polymerase II or RNA polymerase III; show differential effects at TATA-containing vs TATA-less promoters; fail to support cell growth but retain RNA polymerase II function. The resulting mutants will be characterized biochemically for TATA-binding activity, interactions with TFIIA, TFIIB (and potentially other proteins), and by in vitro transcription. Second, we will define gene products, either by suppressor mutations or by overexpression, that revert phenotypes conferred by defective TFIID derivatives. We will use the """"""""two-hybrid"""""""" method for detecting protein-protein interactions to search (as well as test) for TFIID-interacting proteins and to map the interacting surfaces. The genes and the encoded proteins will be characterized by standard techniques of yeast molecular biology. Third, having isolated recessive and dominant suppressor mutations that increase transcription by GCN4 derivatives with partially defective acidic activation regions, we will carry out detailed allele specificity experiments and clone the encoding genes. The goals are to identify proteins that are involved either in the mechanism by which acidic activation domains stimulate the basic transcription machinery or in regulation of GCN4 activity. Fourth, to understand the biological functions of the yeast ATF/CREB protein family, we will perform a mutational analysis of ACR1, identify and characterize ATF/CREB activators, and develop a novel method to identify target genes that are directly regulated by the various ATF/CREB proteins in vivo. Fifth, to determine the mechanisms underlying the functional distinctions between the his3 TATA elements, we will investigate the effect of different activator proteins, the quality of the acidic activation domain, the length of the poly(dA).poly(dT) element and other sequences that affect chromatin structure, competition between TATA elements, and mutations in already identified genes that affect transcription. In addition, we will isolate chromosomal mutations that cause differential effects on transcription mediated by these TATA elements. Sixth, the biochemical functions of the various proteins and protein variants will be examined by in vitro transcription on appropriate promoters using yeast nuclear extracts from wild-type or mutant strains, extracts that have been depleted for TFIID (or other factors), and purified general transcription factors. In addition, protein-protein interaction studies will be carried out by employing standard band-shift assays and affinity chromatography. Seventh, we will obtain new GCN4 derivatives that have altered DNA-binding specificity and design GCN4 mutants and target sites to test specific hypotheses that arise from the X-ray structure of the protein-DNA complex.
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