REGULATION OF EUKARYOTIC RIBOSOMAL RNA TRANSCRIPTION

在细胞中发现的最丰富的RNA种类是核糖体RNA (RRNA)。在动物细胞中，rrna是在特殊密度下合成的。 核仁小体，存在于大多数真核生物的细胞核中。 45S前体rrna的转录是由核仁rna进行的。 聚合酶I和成熟的18S和28S核糖体RNA是在 初级转录产物的转录后处理。这很好 证实了rRNA转录的速度对 环境和细胞生理状态的变化。 尽管rRNA在维持生活状态方面很重要，但几乎没有 已知调节rRNA的分子基础 抄写。在这里，我们建议研究rrna的控制。 在人类细胞中的合成。我们将使用一种无细胞的RNA聚合酶I 从HeLa细胞衍生的转录系统来研究其调控 人核糖体RNA合成。首先，我们将定义启动子区域 通过使用一种新的定点突变来映射控制序列 产生插入、缺失和聚集点突变的程序。 接下来，我们将尝试分离和鉴定特定的转录 赋予RNA聚合酶I启动子选择性所需的因子。 将通过对无细胞生物化学分级来完成 含RNA聚合酶I的转录系统和特异性转录 各种因素。我们还将通过以下方式确定这些转录因子是如何工作的 研究它们结合特定启动子序列和 与RNA聚合酶I亚基的潜在相互作用。最后，我们 将研究核糖体比率变化的机制 细胞对特定代谢和生理反应时的RNA合成 病毒感染带来的变化。

The most abundant species of RNAs found in cells are the ribosomal RNAs (rRNA). In animal cells, rRNAs are synthesized in specialized dense nucleolar bodies which are found in the nucleus of most eukaryotes. Transcription of 45S precursor rRNA is carried out by the nucleolar RNA polymerase I and the mature 18S and 28S ribosomal RNAs are produced after post-transcriptional processing of the primary transcripts. It is well established that the rate of rRNA transcription is acutely sensitive to changes in the environment and to the physiological state of the cell. Despite the importance of rRNA in maintaining the living state, little is known about the molecular basis underlying the regulation of rRNA transcription. Here, we propose to investigate the control of rRNA synthesis in human cells. We will use a cell-free RNA polymerase I transcription system derived from HeLa cells to study the regulation of human ribosomal RNAs synthesis. First, we will define the promoter region by mapping the control sequences using a novel site-directed mutagenesis procedure to generate insertion, deletion, and clustered point mutations. Next, we will attempt to isolate and identify specific transcription factors required to impart promoter selectivity to RNA polymerase I. This will be accomplished by biochemical fractionation of the cell-free transcription system with RNA polymerase I and specific transcription factors. We will also determine how these transcription factors operate by investigating their ability to bind specific promoter sequences and potential interactions with the RNA polymerase I subunits. Finally, we will investigate the mechanism governing changes in the rate of ribosomal RNA synthesis when cells respond to specific metabolic and physiologic changes brought about by viral infection.