The general transcription factors are a class of proteins required for the expression of all genes in the cell. Although much is known about their structure and function, little is known about how their expression might be controlled. This project will pursue the observation made by this laboratory that there are two discrete transcripts that encode the general transcription factor SUA7 (TFIIB) in the yeast Saccharomyces cerevisiae that differ in the amount of 3' untranslated sequence. It was also found that the relative amounts of these two transcripts differ under different cell growth conditions. This research will address how this observation relates to the function of this essential transcription factor in the cell. First, deletion mutagenesis will be performed to create plasmid borne copies of SUA7 that individually affect the accumulation of the two transcripts. Mutations that decrease the formation of the shorter and longer RNA separately will be used in a "plasmid shuffle" assay to allow assessment of the effect of eliminating one or the other of the transcripts on the production of TFIIB protein and on cell growth under different conditions. Second, experiments will be performed to determine the mechanism by which the two transcripts are differentially affected by cellular conditions. A likely hypothesis is that the two transcripts differ in stability and show changes in stability under different growth conditions. If effects on stability are not observed, changes in 3' end processing will be pursued as a possible mechanism for the switch. Third, experiments will be done to address the means by which total SUA7 transcript levels decrease in response to nutrient limitation or amino acid starvation. Since this effect is similar to one seen for ribosomal protein genes, an Abf1p activator binding site in the promoter will be eliminated and altered promoters tested for function in vivo. Elucidating how the DNA sequence of a gene determines its regulation is of critical importance for understanding how an organism develops from a single cell. Determining how the general transcription factors are regulated is an interesting problem, since this class of proteins is itself required for the expression of all genes in the cell. How the levels of these proteins change in response to cell states could potentially have global effects on the production of different protein products. This research seeks to better understand the effect of different cellular conditions on the production of one of these general transcription factors, TFIIB. This particular factor is important in the first steps of the production of RNA from DNA, and several of the characteristics of the gene suggest that learning more about the control of its expression will increase understanding of the effect of cellular conditions on other important classes of genes in the cell.
