GENE SELECTION BY MULTIPLE EUKARYOTIC RNA POLYMERASES

这是一项继续研究启动机制的建议， 真核生物核糖体RNA转录的调控。 有机体是 研究，卡氏阿米巴，是一个很好的模型， 真核生物转录使用生化方法，和显着 上一个供资期间的调查结果合乎逻辑地导致拟议的 实验 具体地说，已经表明DNA依赖性RNA 聚合酶I（pol I）与启动子结合，而不考虑DNA 在pol I结合位点的序列，通过蛋白质-蛋白质相互作用， 转录起始因子（TIF），一种与特定的 核心启动子中的DNA序列。 然而，其中一些复合物与 结合pol I下改变的序列不能启动 转录。 计划进行实验以确定为什么会发生这种情况， 最初的假设是对熔化的顺序效应。 融化已经 已被证明是引发过程中的一个单独步骤， 核苷酸底物的存在。 这有能量要求吗 一步？ 是否有必要形成磷酸二酯键以稳定熔融， 发生，或者核苷酸与聚合酶的结合是否足够？ 的确切时间 是否会发生碱基熔化，以及到底有多少碱基对被熔化？ 如何剂量 DNA超螺旋影响解链和启动子效率？ 角色，如果有的话， 所谓的非转录间隔区（NTS）在促进或增强 将评价rRNa转录。 实验包括测序和 NTS的足迹，NTS与基因启动子的竞争分析 和分析多个TIF结合位点上游的作用， 启动子 后者还应使人们深入了解具体的 转录终止位点，可能还有pol I增强子。 的 TIF和pol I与启动子DNA结合的机制将通过以下详细描述： 进行预修饰足迹技术。 疑问待 解决的问题包括：TIF的绑定常数是什么， 聚合酶？ DNA会被TIF弯曲吗？ 什么是磷酸盐触点？ 什么是糖接触？ TIF结合是否影响DNA构象 RNA聚合酶结合区 如上所述，阿米巴pol I 仅通过与TIF的蛋白质-蛋白质相互作用与rRNA启动子结合。 这种蛋白质-蛋白质相互作用将通过化学和 光化学交联研究。 为了全面落实这一 分析，pol I亚基将被克隆，并影响定点 通过新技术分析诱变。 该实验室证明， 核糖体RNA转录在真核细胞中受 修改poli。 改性的效果是防止聚合物I 与启动子结合，并由此推断，修饰影响启动子的表达。 TIF与聚合酶之间的相互作用。 初步证据显示， 修饰发生在pol I的39 KD亚基上。 实验来 将这种修饰（或后来发现的其他修饰）与调节联系起来， 计划好了 将确定改性的化学性质。 的 进行修饰的酶将被鉴定，并且其机制 对细胞生长率的反应。 除了其 作为研究转录机制的模型系统， 阿米巴是阿米巴角膜炎的病原体， 目前难以治疗与接触透镜相关的角膜感染 穿，贷款直接健康相关的这个项目。

This is a proposal to continue studies of the mechanism of initiation and regulation of eukaryotic ribosomal RNA transcription. The organism being studied, Acanthamoeba castellanii, is an excellent model for the study of eukaryotic transcription using biochemical approaches, and significant findings during the last funding period lead logically into the proposed experiments. Specifically, it has been shown that DNA-dependent RNA polymerase I (pol I) binds to the promoter, without regard to the DNA sequence in the pol I binding site, by protein-protein interaction with transcription initiation factor (TIF), a protein which binds to a specific DNA sequence in the core promoter. Yet, some of these complexes with altered sequence under the bound pol I are unable to initiate transcription. Experiments are planned to determine why this occurs, with the initial hypothesis being a sequence effect on melting. Melting has been shown to be a separate step in the initiation process requiring the presence of nucleotide substrates. Is there an energy requirement for this step? Is it necessary to form phosphodiester bonds for stable melting to occur, or is nucleotide binding to the polymerase sufficient? Exactly when does melting occur, and exactly how many base pairs are melted? How dose DNA supercoiling affect melting and promoter efficiency? The role, if any, of the so called non-transcribed spacer (NTS) in promoting or enhancing rRNa transcription will be evaluated. Experiments include sequencing and footprinting of the NTS, analysis of NTS competition with the gene promoter and analysis of the effect of multiple TIF binding sites upstream of the promoter. The latter should also give an insight into the role of specific sites of transcription termination, and perhaps pol I enhancers. The mechanism of binding of TIF and pol I to promoter DNA will be detailed by carrying out premodification footprinting techniques. Questions to be addressed include: What is the binding constant for TIF, and for polymerase? Is the DNA bent by TIF? What are the phosphate contacts? What are the sugar contacts? Does TIF binding affect DNA conformation in the RNA polymerase binding region? As mentioned above, Acanthamoeba pol I binds to the rRNA promoter solely by protein-protein interactions with TIF. This protein-protein interaction will be analyzed by chemical and photochemical cross-linking studies. In order to fully carry out this analysis, pol I subunits will be cloned, and the effect of site-directed mutagenesis analyzed by novel techniques. This laboratory demonstrated that ribosomal RNA transcription is regulated in eukaryotic cells by modification of pol I. The effect of modification is to prevent pol I from binding to the promoter, and by inference, modification affects the interaction between TIF and the polymerase. Preliminary evidence shows a modification to occur on the 39KD subunit of pol I. Experiments to correlate this modification (or others found later) with regulation are planned. The chemical nature of the modification will be determined. The enzyme which carries out modification will be identified, and its mechanism of response to cellular growth rate investigated. In addition to its serving as a model system for studies of transcription mechanisms, Acanthamoeba is the causative agent of amoebic keratitis, a serious and currently difficult to treat corneal infection associated with contact lens wear, lending direct health relevance to this project.