RNA POLYMERASE III TRANSCRIPTION REGULATION MECHANISM

5S RNA和核糖体RNA的转录速率以及核糖体的表达 蛋白质与细胞的生长速度密切相关，以便平衡 核糖体的精加工。这项建议旨在研究 协调这些基因表达的机制，这些基因是 由不同的RNA聚合酶转录。在棘阿米巴中，rRNA 转录受聚合酶I修饰调控，最近 结果表明，5S RNA转录关闭伴随着 核内TFIIlA转录活性和DNA结合活性丧失。这个 这种损失的机制尚不清楚。这项研究将考察这一变化 几种方法检测TFIIlA活性：TFIIlA蛋白水平 Western印迹分析将揭示整个细胞中是否存在该蛋白。一个 变化将表明细胞中的TFIIlA较少。如果是这样的话，一个 对这一变化机制的研究将涉及克隆 TFIIlA基因及TFIIlA mRNA是否平行下降的判定 含有TFIIlA蛋白。如果是这样，核径流实验将确定 TFIIlA基因水平是否受转录调控。如果TFIIlA 是转录调控的，对其机制的研究 这一规定将付诸实施。第一，克隆人的启动子 TFIIlA基因将通过体外转录进行解剖。如果TFIIlA 转录水平不受调控，TFIIlA与mRNA相关 将对多聚体进行检查以确定翻译是否 尤其是受到了阻碍。或者，如果整个细胞水平 TFIIlA的细胞定位保持不变，例如。作为一名 细胞质中含有5S RNA的复合体，将通过分析 TFIIlA在细胞核和细胞质中的分布，并通过 7S RNP和42S RNP在细胞质中的含量测定 细胞发育的不同阶段与5S RNA转录 活动。如果细胞核中存在的TFIIIA蛋白的数量是恒定的， 但只有该因子的DNA结合活性发生改变，然后TFIIIA 必须进行修饰，使其不能与5S RNA基因相互作用。一个 对修改的调查将由结构 活性细胞和非活性细胞TFIIlA的比较。晚些时候 棘阿米巴发育，TFIIIC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来研究这种变化的机制 对TFIIIA的上述研究。

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.