RNA POLYMERASE I TRANSCRIPTION AND REGULATION MECHANISMS

描述：这是一个继续研究真核生物的建议。 核糖体RNA转录起始和调节。 RNA聚合酶 I（pol I）转录该基因。 真核生物的rRNA基因 正在研究的生物，阿米巴，具有使其 特别适合于建议的生物化学研究;性能不 目前，该系统还在研发中。 特别是， 其核心启动子和转录之间的复合物的稳定性 因子允许通过其他系统中不可用的技术进行分析。 最近的调查结果合乎逻辑地导致这一建议。 整个rRNA 基因间间隔区测序，pol I增强子鉴定， 显示结合脊椎动物UBF同源物。 TATA结合蛋白（TBP） 含有起始因子（TIF-IB）的细胞被纯化至均一， 亚基（TAFIS）鉴定，TBP在pol I中的独特作用 转录显示。 NTP β-γ水解被证明不是 启动所需的，并通过聚合酶I的DNA解链过程进行了检查 详细 TIF-IB和pol I的位点直接光交联， 启动子DNA导致TAFIS和TBP的拓扑结构在 rRNA启动子。 再加上STEM图片， 到激活的启动子复合体从未详细，并给出了 对pol I启动子如何发挥作用有重要的见解。 监管 研究表明pol α亚基同源物（AC 39）参与调节， 并对其进行了克隆和测序。 基金目前 要求达到以下目标：（A）蛋白质和蛋白质- DNA相互作用将进一步详细使用新的复杂的 光亲和探针，异双功能交联剂，和高 解析STEM。 (B)几个潜在的额外的作用，如果有的话， 影响rRNA转录的转录因子， 评估。 其中包括一个类似Ku的因子和一个 在核心启动子上形成稳定的复合物。 (C)监管 rRNA的转录机制将进行研究，重点放在 对导致纯化的聚合酶I失活的化学事件的研究 来自转录不活跃的细胞。 调查已知的 AC 39亚基的修饰及其在调控中的作用，将被 一个特定的目标。 (D)TAFIS和关键pol I亚基的基因将 被克隆。 (E)提出了一种将非线性系统的暂态和稳态变换为线性系统的方法， 会产生阿米巴原虫。

DESCRIPTION: This is a proposal to continue studies of eukaryotic ribosomal RNA transcription initiation and regulation. RNA polymerase I (pol I) transcribes this gene. The rRNA gene of the eukaryotic organism being studied, Acanthamoeba, has properties making it especially good for the biochemical studies proposed; properties no other pol I system currently exhibits. In particular, the unusual stability of the complex between its core promoter and transcription factor allow analysis by techniques not available in other systems. Recent findings lead logically into this proposal. The entire rRNA intergenic spacer was sequenced, pol I enhancers were identified, and shown to bind a vertebrate UBF homolog. The TATA-binding protein (TBP)- containing initiation factor (TIF-IB) was purified to homogeneity, its subunits (TAFIS) identified, and TBP's distinctive role in pol I transcription revealed. NTP beta-gamma hydrolysis was shown not to be needed for initiation, and the DNA melting process by pol I was examined in detail. Site-direct photocross-linking of TIF-IB and pol I to promoter DNA resulted in mapping the topology of TAFIS and TBP on the rRNA promoter. Coupled with STEM pictures, this study has given insights into the activated promoter complex never before detailed, and gave significant insights into how pol I promoters function. Regulatory studies implicated the pol alpha subunit homolog (AC39) in regulation, and inroads into its cloning and sequencing were made. Funds are now requested to attain the following aims: (A) Protein-protein and protein- DNA interaction will further detailed using new sophisticated photoaffinity probes, heterobifunctionals cross-linking agents, and high resolution STEM. (B) The role, if any, of several potential additional transcription factors which influence rRNA transcription will be evaluated. These include a Ku-like factor and a factor required for stable complex formation on the core promoter. (C) The regulatory mechanism of rRNA transcription will be investigated, placing emphasis on the chemical events resulting in the inactivity of pol I purified from transcriptionally inactive cells. Investigation of the known modification of the AC39 subunits, and its role in regulation, will be a particular aim. (D) Genes for the TAFIS and key pol I subunits will be cloned. (E) A method for transient and stable transformation of Acanthamoeba will be developed.