RNA POLYMERASE I TRANSCRIPTION AND REGULATION MECHANISMS

描述：这是一项继续研究真核生物的建议 核糖体RNA转录的启动和调控。RNA聚合酶 我(PolI)转录这个基因。真核生物的rRNA基因 正在研究的生物体棘阿米巴具有使其 特别适合建议的生物化学研究；特性不 1号刑警组织的其他系统目前正在展出。特别是，不同寻常的 其核心启动子与转录之间的复合体的稳定性 因子允许使用其他系统所不具备的技术进行分析。 最近的发现顺理成章地引出了这一提议。整个rRNA 对基因间隔区进行测序，鉴定PolI增强子，并 显示与脊椎动物的UBF同源物结合。塔塔结合蛋白(TBP)- 含启动因子(TIF-IB)纯化至均一，其 确定的亚基(TAFI)，以及TBP在POL I中的独特作用 文字记录显示。NTPβ-伽马水解酶被证明不是 需要启动，并检测了PolI的DNA熔化过程 详细地说。TIF-IB和POL I的原位直接光交联 启动子DNA将TAFIS和TBP的拓扑结构映射到 RRNA启动子。结合STEM图片，这项研究给出了见解 对激活的启动子复合体进行了前所未有的详细介绍，并给出了 对POLI启动子如何发挥作用的重大见解。监管 研究表明，Pola亚基同源基因(AC39)参与调控， 并在其克隆和测序方面取得了进展。基金现在是 要求达到以下目标：(A)蛋白质--蛋白质和蛋白质-- DNA相互作用将使用新的复杂技术进一步详细说明 光亲和力探针、异双官能团交联剂和高 决议STEM。(B)几个潜在的额外成员的作用(如有的话) 影响rRNA转录的转录因子将是 已评估。它们包括类Ku因子和所需的因子 在核心启动子上形成稳定的复合体。(C)监管机构 将研究rRNA转录的机制，重点是 关于导致PolI纯化失活的化学事件 来自转录不活跃的细胞。对已知事件的调查 AC39亚基的修饰及其在调节中的作用将是 一个特定的目标。(D)TAFIS和主要PolI亚基的基因将 被克隆。(E)一种暂态和稳定转变的方法 将开发棘阿米巴。

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.