Molecular mechanisms of transcriptional regulation
The focus of research in our laboratory is to understand the molecular mechanisms of eukaryotic transcription initiation. For the last ten or so years we have been examining the eukaryotic transcription factors which mediate initiation complex formation and thus represent potential targets for trans-regulation. We have utilized the simple eukaryote, Saccharomyces cerevisiae or Baker's Yeast, for our work. This organism was chosen for our studies because both biochemical and genetic approaches can be taken with yeasts. In our experiments we study the factors required for transcription initiation by RNA polymerase II (RNAP II). RNAP II transcribes the genes which encode mRNAs. We have developed methods for the solubilization, characterization and purification of the complete complement of RNAP II-specific factors, and our current focus is on one of these factors the multisubunit factor termed TFIID. All of the proteins which comprise TFIID have very interesting biochemical properties. One of the factors, known as TBP, or TATA box Binding Protein, is a sequence specific DNA binding protein which interacts with the ubiquitous TATA box promoter element. while others are not. The other subunits of TFIID presumably interact with other promoter elements, RNAPII, positive-acting transcription factors or other general transcription factors such as TFIIA, TFIIB, TFIIE, TFIIF or TFIIF.
Our immediate efforts have been expended towards cloning the yeast genes which encode these genes encoding the TFIID subunits. We have been successful in cloning the genes which encode the multiple (15 distinct genes) subunits of yeast TFIID. Our interest in cloning these genes are several and are summarized here as are the types of studies which will be the focus of our research in the future--each could comprise a student rotation project: 1) The cloned genes give us the wherewithal to overexpress the corresponding gene products. Purified factors prepared from the cloned genes will be used for in vitro mechanistic studies. 2) Using the cloned genes we are examining the structure-function relationships of these important molecules. 3) We are dissecting the genetic control elements which regulate expression of the transcription factor genes themselves. These studies are being performed with an eye towards understanding global control of macromolecular biosynthesis. 4) Finally, we are using the cloned genes, in conditionally lethal forms, to identify via suppressor analyses genes whose products interact with these multi-functional general transcription initiation factors. Specific examples of the types of studies and the results which we have obtained are listed in "Selected Publications."