REGULATION OF EUKARYOTIC RIBOSOMAL RNA TRANSCRIPTION

细胞中最丰富的RNA是核糖体RNA （rRNA）。 在动物细胞中，rRNAs是在专门的致密的 在大多数真核生物的细胞核中发现的核仁体。 45S rRNA前体的转录是由核仁RNA进行的。 聚合酶I和成熟的18S和28S核糖体RNA在 初级转录物的转录后加工。 公 确定rRNA转录速率对以下因素非常敏感： 环境和细胞生理状态的变化。 尽管rRNA在维持生命状态方面很重要， 已知rRNA调控的分子基础 转录。 在这里，我们建议研究rRNA的控制 在人体细胞中合成。 我们将使用无细胞RNA聚合酶I 从HeLa细胞中获得的转录系统，以研究 人核糖体RNA合成。 首先，我们将定义启动子区域 通过使用新的定点诱变来定位控制序列， 产生插入、缺失和成簇点突变的程序。 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。 这 将通过无细胞的生物化学分级分离来完成 具有RNA聚合酶I和特异性转录的转录系统 因素 我们还将确定这些转录因子如何运作， 研究它们结合特异性启动子序列的能力， 与RNA聚合酶I亚基的潜在相互作用。 最后我们 将研究核糖体的速率变化的机制， 当细胞对特定的代谢和生理反应作出反应时， 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.