RNA POLYMERASE III TRANSCRIPTION REGULATION MECHANISM

5S RNA和核糖体RNA的转录速率，以及核糖体RNA的表达。 蛋白质与细胞生长速度密切相关， 核糖体的加工。这项建议旨在研究 协调这些基因表达的机制， 由不同的RNA聚合酶转录。在阿米巴中，rRNA 转录受聚合酶I修饰调节，最近 结果表明，5S RNA转录关闭伴随着一个 细胞核中TFIlA转录和DNA结合活性的丧失。的 这种损失的机制尚不清楚。本研究将探讨 通过几种方法测定TFIlIA活性： 蛋白质印迹分析将揭示整个细胞中存在的蛋白质。一 这种变化将指示细胞中存在较少的TFIlA。如果是， 对这种变化机制的研究将涉及克隆 TFIIlA的基因，并确定TFIIlA mRNA是否平行下降 TFIlA蛋白。如果是这样，核泄漏实验将决定 TFIII A mRNA水平是否受转录调控。如果TFIIlA 是转录调控的，一项对转录调控机制的研究， 这项规定将得到执行。首先，克隆的启动子 将使用体外转录来剖析TFIlA基因。如果TFIIlA mRNA水平不受转录调节，TFIlA mRNA相关性 将检查带有多聚核糖体的蛋白质，以确定翻译是否正确 具体受阻。或者，如果总的细胞水平 TFIlA保持恒定，TFIlA的细胞定位，例如。作为 与细胞质中的5S RNA复合物，将通过分析 TFIlA在细胞核和细胞质中的分布，以及 测定细胞质中7S RNP和42S RNP的量， 细胞发育的不同阶段和5S RNA转录 活动如果存在于细胞核中的TFIIIA蛋白的量是恒定的， 但只有该因子的DNA结合活性改变，则TFIIIA 必须进行修饰，使其不能与5S RNA基因相互作用。一个 修改调查将由结构 来自活性和非活性细胞的TFIlA的比较。晚些 阿米巴发育，TFIIIC的活性水平降低， 推测是为了关闭其他2型聚合酶III的转录。我们 我将使用类似的方法研究这种变化的机制， 上述研究的TFIIIA。

5S RNA and ribosomal RNA transcription rates, and expression of ribosomal proteins are closely tied to cellular growth rate in order to balance elaboration of ribosomes. This proposal is aimed at studying the mechanism of coordinating expression from these genes, which are transcribed by distinct RNA polymerases. In Acanthamoeba, rRNA transcription is regulated by polymerase I modification, and recent results show that 5S RNA transcriptional shutdown is accompanied by a loss in TFIIlA transcriptional and DNA binding activity in nuclei. The mechanism of this loss is unknown. This study will examine the change in TFIIlA activity by several approaches: The level of TFIIlA protein present in the whole cell will be revealed by Western blot analysis. A change would indicate that there is less TFIIlA in the cell. If so, an investigation of the mechanism for this change will involve cloning the gene for TFIIlA and determining whether TFIIlA mRNA declines in parallel with TFIIlA protein. If so, nuclear run off experiments will determine whether the TFIIlA mRNA level is regulated transcriptionally. If TFIIlA is regulated transcriptionally, an investigation into the mechanism of this regulation will be carried out. First, the promoter of the cloned TFIIlA gene will be dissected using in vitro transcription. If TFIIlA mRNA levels are not regulated transcriptionally, TFIIlA mRNA association with polysomes will be examined to determine if translation is specifically hindered. Alternatively, if the overall cellular level of TFIIlA remains constant, the cellular localization of TFIIlA, eg. as a complex with 5S RNA in the cytoplasm, will be investigated by analyzing the distribution of TFIIlA in nuclei and the cytoplasm, and by determining the amount of 7S RNP and 42S RNP in the cytoplasm at different stages of cellular development and 5S RNA transcriptional activity. If the amount of TFIIIA protein present in nuclei is constant, but only the DNA binding activity of the factor is altered, then TFIIIA must be modified so that it cannot interact with the 5S RNA gene. An investigation of modification will be carried out by structural comparison of TFIIlA from active and inactive cells. Later in Acanthamoeba development, the activity level of TFIIIC is reduced, presumably to shut down other type 2 polymerase III transcription. We will investigate the mechanism of this change using an approach similar to the above study of TFIIIA.