RUI: Regulation of the Expression of the TFIIB Gene of Saccharomyces cerevisiae

一般转录因子是细胞中所有基因表达所必需的一类蛋白质。虽然人们对它们的结构和功能了解很多，但对它们的表达可能如何控制却知之甚少。本项目将继续本实验室所做的观察，即在酿酒酵母中存在两个编码通用转录因子SUA7(TFIIB)的离散转录本，其3‘非翻译序列的数量不同。研究还发现，在不同的细胞生长条件下，这两种转录本的相对数量不同。这项研究将解决这种观察如何与细胞中这种基本转录因子的功能有关。首先，将进行缺失突变，以产生分别影响两个转录本积累的质粒型SUA7副本。分别减少较短和较长RNA形成的突变将用于“质粒改组”试验，以评估在不同条件下消除其中一个转录本对TFIIB蛋白的产生和对细胞生长的影响。其次，将进行实验以确定这两个转录本受细胞条件影响的不同机制。一个可能的假设是，这两个转录本的稳定性不同，并且在不同的生长条件下显示出稳定性的变化。如果没有观察到对稳定性的影响，将寻求3‘端加工的变化作为切换的可能机制。第三，将进行实验，以解决营养限制或氨基酸饥饿导致SUA7总转录水平下降的方法。由于这种效应类似于核糖体蛋白基因，启动子中的Abf1p激活子结合位点将被消除，并改变启动子在活体中的功能测试。了解基因的DNA序列如何决定其调节对于理解生物如何从单个细胞发育至关重要。确定一般转录因子是如何调控的是一个有趣的问题，因为这类蛋白质本身是细胞中所有基因表达所必需的。这些蛋白质的水平如何随着细胞状态的变化而变化，可能会对不同蛋白质产品的生产产生全球影响。这项研究旨在更好地了解不同细胞条件对这些通用转录因子之一TFIIB产生的影响。这个特殊的因素在从DNA产生RNA的第一步中很重要，该基因的几个特征表明，更多地了解其表达的控制将增加对细胞条件对细胞中其他重要类别基因的影响的理解。

9809917HoopesThe 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.