REGULATION OF EUKARYOTIC RIBOSOMAL RNA TRANSCRIPTION

我们的主要目标是识别、分离和确定 核糖体RNA(RRNA)转录系统中的组件，因此 管理RNA合成的调节机制可以被操纵和 在生化水平上进行分析。我们之前开发了一种 精准启动RNA合成的体外分步系统 人类核糖体启动子，我们已经定义了顺式调控 指定启动子功能的序列。此外，我们最近已经 鉴定和部分纯化物种特异性启动子识别 因子，称为SLL。最重要的是，初步的DNA结合研究和 定点突变分析表明，SLL与 另一个转录成分，可能是RNA聚合酶I本身，形成 然后可以识别上游控件并与其交互的复合体 启动者的要素。在这项续期拨款中，我们建议分析 蛋白质-DNA和蛋白质-蛋白质相互作用的性质和特异性 它们发生在SL1、RNA聚合酶I和启动子序列之间。这些 生化研究应该为我们提供关于 哺乳动物转录激活的分子机制 RRNA基因。第一，各种DNA结合实验，包括DNA酶 足迹法、Carthers凝胶结合试验、DMS甲基化保护、MPE 利用野生型和突变体进行保护和烷基化干扰 模板将被用来分析蛋白质-DNA的相互作用， 启动子识别。下一步，我们计划净化SLL以及其他 与SLL形成复合体并产生单抗的蛋白质 针对这些转录因子。如果高滴度抗体可以 ，然后我们将使用它们来帮助我们识别和表征 SLL多肽及其与其他相关多肽的相互作用 在转录起始反应中。抗体也可以用来 为了检测和定量转录因子在细胞中的存在 不同生长和环境胁迫下细胞的核仁 条件，以便将转录激活与 存在特定的反式激活因子。在获得了高度的 纯化和活性的SLL，我们将尝试微量注射纯化的因子 转化成各种类型的细胞，如人-鼠嵌合体，以测试这一想法 SLL可能是哺乳动物细胞核仁优势的原因 混血儿。最后，我们将尝试克隆和鉴定这些基因 编码SLL和其他相关转录因子，目的是 最终开发出可以过度生产一个或多个 这些低丰度转录蛋白。

Our main objective is to identify, isolate, and determine the function of components in the ribosomal RNA (rRNA) transcription system, so that the regulatory mechanisms that govern RNA synthesis can be manipulated and analyzed at the biochemical level. We had previously developed a fractionated in vitro system that accurately initiates RNA synthesis from the human ribosomal promoter, and we had defined the cis regulatory sequences that specify promoter function. In addition, we have recently identified and partially purified a species-specific promoter recognition factor, called SLl. Most importantly, preliminary DNA binding studies and site-directed mutagenesis analysis suggests that SLl, in conjunction with another transcriptional component, possibly RNA polymerase I itself, forms a complex that can then recognize and interact with the upstream control element of the promoter. In this renewal grant, we propose to analyze the nature and specificity of the protein-DNA and protein-protein interactions that occur between SL1,RNA polymerase I and promoter sequences. These biochemical studies should provide us with new insights concerning the molecular mechanism that mediates transcriptional activation of mammalian rRNA genes. First, a variety of DNA binding experiments, including DNAse footprinting, Carothers gel binding assays, DMS methylation protection, MPE protection, and alkylation-interference using both wild-type and mutant templates will be used to analyze the protein-DNA interactions that direct promoter recognition. Next, we plan to purify SLl as well as other proteins that form a complex with SLl and raise monoclonal antibodies directed against these transcription factors. If high titer antibodies can be obtained, we will then use them to help us identify and characterize the SLl polypeptide as well as its interaction with other polypeptides involved in the transcription initiation reaction. The antibodies can also be used to detect and quantitate the presence of transcription factors in the nucleolus of cells under different growth and environmental stress conditions in order to correlate transcriptional activation with the presence of specifc trans-activating factors. After obtaining highly purified and active SLl, we will attempt to microinject the purified factor into a variety of cell types such as human-mouse chimeras, to test the idea that SLl may be responsible for nucleolar dominance in mammalian-cell hybrids. Finally, we will attempt to clone and characterize the genes encoding SLl and other relevant transcription factors with the aim of eventually developing expression vectors that can overproduce one or more of these low abundance transcription proteins.