Carl S. Parker - US grants

The research described in this proposal is directed at identifying and purifying general RNA polymerase II transcription factors. We are currently purifying one of the identified factors by virtue of its DNA binding affinity. This factor was identified by us in a Drosophila cultured cell nuclear extract, and found to bind to the TATA-homology element present on a wide variety of RNA polymerase II genes. Since that time we have gained extensive knowledge of the chromatographic properties of this factor (designated the B factor). Using conventional chromatography we have not been able to purify the B factor to homogeneity. This problem was also encountered during the purification of the Drosophila heat shock transcription factor (HSTF). The HSTF was recently successfully purified using sequence- specific DNA affinity chromatography. We are currently in the process of using the same technique to purify the B factor from Drosophila and human cells (HeLa cells). We are systematically testing various promoter sequences to identify the best sequence to use for the chromatography. Preliminary information with one promoter sequence has already yielded promising results and is described in the proposal. Once the B factor has been purified to homogeneity we plan to obtain the gene for this protein by using two approaches. We will obtain an N-terminal sequence from a fragment using an Applied Biosystems protein sequenator. From the DNA sequence the appropriate oligonucleotide will be synthesized to probe a Drosophila cDNA library. The second approach planned is to obtain antibodies antibodies against the protein and screen a Drosophila cDNA library cloned into lambda gt11. Using purified B factor we plan to perform a number of experiments to characterize its interactions with DNA including alkylation-interference, and localized-denaturation determination. With the cloned B factor gene a large number of experiments are envisioned including DNA binding domain identification as well as site-directed mutagenesis.

We plan on continuing our analysis of the mechanism by which the segmentation gene fushi tarazu (ftz) is temporally and spatially regulated at the level of transcription. Our past work has focused on the cis acting control elements present in the zebra promoter element. We found that two regulatory sites when present as an array of individual oligonucleotide binding sites were capable of reconstructing the striped pattern of ftz transcription in the embryo. These two regulatory elements have thus become the focus of our current work and this continuation proposal. One of these two sites is termed the dual element (DE) because it provides both an activation function as well as a repressor function. The other site is termed repressor element-1 (RE-1) because it seems to provide only a repressor function. We have cloned and partially characterized two genes whose products bind to the DE. They are both novel members of the steroid receptor super- family, and possess a unique amino acid sequence in their DNA binding domain. The proteins that we have identified which bind to the RE-1 include the giant protein and a novel gene product which contains a basic region than is only now being characterized. In the future we plan to carry out a detailed analysis of these and other potential regulators of ftz transcription to understand the mechanism by which they function to control transcription.

The specific aims of this proposal are to gain a molecular understanding of how the heat shock transcription factor (HSF) is activated by heat shock, and to determine how activation of the factor occurs. We plan on focusing on the signal transduction pathway that modulates the activity of the HSF from Drosophila and yeast. We will determine the residues of HSF that are phosphorylated in normally growing cells, heat shocked cells and during recovery from heat shock. We will generate point mutations in the phosphorylated residues of the HSFs and determine whether they are regulatory in vivo. We will identify and isolate kinases present in extracts prepared from non-shocked, shocked and recovering cells and compare their phosphorylation pattern with that of HSF in vivo. Those kinases which generate a 2D phosphopeptide map pattern similar to or overlapping with the in vivo map will be purified and characterized. It is likely that these kinases play an important role in the signal transduction of the stress response. Drosophila and S. cerevisiae HSF's differ in their mode of regulation in that nuclear entry of the Drosophila factor is also regulated by stress. We have identified an important domain of the Drosophila HSF which plays a key role in this process. We propose to identify co-factors which associate with this domain in vivo, purify them, and attempt to determine their mechanism of action.