MECHANISMS OF YEAST TRANSCRIPTIONAL INITITATION

真核生物调节基因表达的机制是 对基本的科学知识很重要，这是 了解许多复杂的生物现象，包括人类疾病。 关于转录启动的过程，有很多不同的 的实验已经指出了真核生物中常见的分子机制 从人类到酵母菌的各种生物。这项提议将继续 探讨与分子机制有关的几个基本问题 在酵母中通过结合多种不同的转录调控 包括重组DNA技术、分子技术和经典技术 酵母遗传学、蛋白质和核酸生物化学。首先，我们 将通过获得突变体来研究TFIID的各种功能 具有以下属性：支持基本转录，但无法 对酸性激活蛋白有反应；是有特定缺陷的 RNA聚合酶II或RNA聚合酶III转录；显示 含有TATA的启动子与不含TTA的启动子的差异效应；未能 支持细胞生长，但保留RNA聚合酶II的功能。由此产生的 突变株的生物化学特征是TATA结合活性， 与TFIIA、TFIIB(以及可能的其他蛋白质)的相互作用，并通过 体外转录。其次，我们将通过以下方式定义基因产品 抑制子突变或通过过度表达，逆转表型 由有缺陷的TFIID衍生品授予。我们将使用“双杂交” 一种检测蛋白质相互作用以进行搜索的方法(以及 测试)寻找与TFIID相互作用的蛋白质，并绘制相互作用表面。 这些基因和编码的蛋白质将通过标准的 酵母分子生物学技术。第三，隔离隐性 和显性抑制突变，通过Gcn4增加转录 具有部分有缺陷的酸性活化区的衍生物，我们将 进行详细的等位基因特异性实验，克隆编码 基因。我们的目标是找出参与其中的蛋白质 酸性活化区刺激碱性的机制 转录机制或GCN4活性的调节。第四，到 了解酵母ATF/CREB蛋白家族的生物学功能， 我们将进行ACR1的突变分析，鉴定和表征 ATF/CREB激活剂，并开发了一种识别靶基因的新方法 在体内受到各种ATF/CREB蛋白的直接调节。 第五，确定功能差异背后的机制 在他的3个塔塔元素之间，我们将调查 不同的激活蛋白，酸性激活的质量不同 结构域、聚(Da)、聚(Dt)元件和其他序列的长度 影响染色质结构，塔塔元件之间的竞争，以及 已经确定的影响转录的基因突变。在……里面 此外，我们将分离导致差异的染色体突变 这些TATA元件对转录的影响。第六， 各种蛋白质和蛋白质变体的生化功能将 在适当的启动子上通过体外转录进行检测 野生型或突变型菌株的酵母核提取物 已耗尽TFIID(或其他因素)，并提纯一般 转录因子。此外，蛋白质-蛋白质相互作用的研究 将通过使用标准的带移分析和亲和力来进行 层析法。第七，我们将获得新的GCN4衍生品 DNA结合特异性改变与Gcn4突变体和靶点的设计 为了测试从X射线结构中产生的特定假设 蛋白质-DNA复合体。

The mechanisms by which eukaryotic organisms regulate gene expression are important for basic scientific knowledge that is necessary for understanding many complex biological phenomena including human diseases. With regard to the process of transcriptional initiation, a wide variety of experiments have pointed to common molecular mechanisms in eukaryotic organisms ranging from humans to yeasts. This proposal will continue to investigate several basic issues concerning the molecular mechanisms of transcriptional regulation in yeast by combining a wide variety of techniques including recombinant DNA technology, molecular and classical yeast genetics, and protein and nucleic acid biochemistry. First, we will investigate the various functions of the TFIID by obtaining mutants with the following properties: support basal transcription but fail to respond to acidic activator proteins; are specifically defective either for transcription by RNA polymerase II or RNA polymerase III; show differential effects at TATA-containing vs TATA-less promoters; fail to support cell growth but retain RNA polymerase II function. The resulting mutants will be characterized biochemically for TATA-binding activity, interactions with TFIIA, TFIIB (and potentially other proteins), and by in vitro transcription. Second, we will define gene products, either by suppressor mutations or by overexpression, that revert phenotypes conferred by defective TFIID derivatives. We will use the "two-hybrid" method for detecting protein-protein interactions to search (as well as test) for TFIID-interacting proteins and to map the interacting surfaces. The genes and the encoded proteins will be characterized by standard techniques of yeast molecular biology. Third, having isolated recessive and dominant suppressor mutations that increase transcription by GCN4 derivatives with partially defective acidic activation regions, we will carry out detailed allele specificity experiments and clone the encoding genes. The goals are to identify proteins that are involved either in the mechanism by which acidic activation domains stimulate the basic transcription machinery or in regulation of GCN4 activity. Fourth, to understand the biological functions of the yeast ATF/CREB protein family, we will perform a mutational analysis of ACR1, identify and characterize ATF/CREB activators, and develop a novel method to identify target genes that are directly regulated by the various ATF/CREB proteins in vivo. Fifth, to determine the mechanisms underlying the functional distinctions between the his3 TATA elements, we will investigate the effect of different activator proteins, the quality of the acidic activation domain, the length of the poly(dA).poly(dT) element and other sequences that affect chromatin structure, competition between TATA elements, and mutations in already identified genes that affect transcription. In addition, we will isolate chromosomal mutations that cause differential effects on transcription mediated by these TATA elements. Sixth, the biochemical functions of the various proteins and protein variants will be examined by in vitro transcription on appropriate promoters using yeast nuclear extracts from wild-type or mutant strains, extracts that have been depleted for TFIID (or other factors), and purified general transcription factors. In addition, protein-protein interaction studies will be carried out by employing standard band-shift assays and affinity chromatography. Seventh, we will obtain new GCN4 derivatives that have altered DNA-binding specificity and design GCN4 mutants and target sites to test specific hypotheses that arise from the X-ray structure of the protein-DNA complex.