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.
| Zhang-Keck, Z Y; Kibbe, W A; Moye-Rowley, W S et al. (1991) The SV40 core sequence functions as a repressor element in yeast. J Biol Chem 266:21362-7 |
| Muhich, M L; Iida, C T; Horikoshi, M et al. (1990) cDNA clone encoding Drosophila transcription factor TFIID. Proc Natl Acad Sci U S A 87:9148-52 |
| Wiederrecht, G; Seto, D; Parker, C S (1988) Isolation of the gene encoding the S. cerevisiae heat shock transcription factor. Cell 54:841-53 |
| Wiederrecht, G; Shuey, D J; Kibbe, W A et al. (1987) The Saccharomyces and Drosophila heat shock transcription factors are identical in size and DNA binding properties. Cell 48:507-15 |
| Shuey, D J; Parker, C S (1986) Bending of promoter DNA on binding of heat shock transcription factor. Nature 323:459-61 |
