MECHANISM OF 5S RNA SYNTHESIS

关于非洲爪哇5S RNA基因转录调控的研究 建立了这个基因系统，作为理解 真核生物转录调控。5S RNA基因通过以下方式激活 一种名为TFIIIA的转录因子与ENTER的结合 120个核苷酸的长基因。这种转录因子，即 锌指DNA结合蛋白的原型具有新的能力 与5S RNA和5S DNA特异性结合。提出了实验方案。 进一步研究含5S的7S粒子的详细结构 RNA和TFIIIA。初步结果导致了7S粒子的模型 将在这些实验中直接测试的结构。约束： TFIIIA对该基因的转录激活只是第一步。 转录因子TFIIIC与A因子-DNA复合体结合形成 稳定的络合物，通过第二个模板抵抗竞争。进一步 将对非洲爪哇TFIIIC进行纯化，以确定其程度 它的两个可分离组件在结构和功能上相似 人类TFIIIC1和TFIIIC2。DNA酶保护实验将 进行以确定非洲爪哇TFIIIC2是否与 转录启动，已被认为是人类的对应物。 TFIIIa间间隔改变的突变5S RNA基因集合 结合部位和起始部位(包含推测的TFIIIC2 结合位点)将被研究来关联转录因子结合和 DNA酶保护稳定复合体。一种新型的蛋白酶保护剂 “足迹”技术将在稳定的综合体中发展。三 计划进行一系列实验来研究RNA聚合酶III的作用。 首先，将采用化学标签程序来识别 RNA聚合酶活性部位的多肽 转录的起始和早期延伸模式。第二， 活性蛋白激酶在RNA聚合酶III中的意义将是 调查过了。最后，关于RNA聚合酶II是否 能够准确终止，而不需要La抗原作为 终止因素将重新调查。

Studies of the control of transcription of Xenopus 5S RNA genes have established this gene system as a leading model for understanding the regulation of transcription in eukaryotes. The 5S RNA gene is activated by the binding of a transcription factor, designated TFIIIA, to the enter of the 120 nucleotide long gene. This transcription factor, which is the prototype for a zinc-finger DNA binding protein, has the novel ability to bind specifically to 5S RNA as well as to 5S DNA. Experiments are proposed to further study the detailed structure of the 7S particle containing 5S RNA and TFIIIA. Preliminary results have led to a model for 7S particle structure that will be directly tested in these experiments. Binding of TFIIIA to the gene is only the first step in transcriptional activation. Transcription factor TFIIIC binds to the factor A-DNA complex to form a stable complex that resists competition by a second template. Further purification of Xenopus TFIIIC will be conducted to determine the extent to which its two separable components structurally and functionally resemble the human TFIIIC1 and TFIIIC2. DNase protection experiments will be conducted to determine whether the Xenopus TFIIIC2 binds to the site of transcription initiation, as has been suggested for its human counterpart. A collection of mutant 5S RNA genes with altered spacing between the TFIIIa binding site and the initiation site (containing the presumed TFIIIC2 binding site) will be studied to correlate transcription factor binding and DNAse protection stable complex. A novel protease-protection "footprinting" technique will be developed in the stable complex. Three series of experiments are planned to study the role of RNA polymerase III. First, a chemical labeling procedure will be adapted to identify the polypeptides in the active site of the RNA polymerase in both the initiation and early elongation modes of transcription. Second, the significance of an active protein kinase in RNA polymerase III will be investigated. Finally, the question of whether RNA polymerase II is capable of accurate termination without the action of La antigen as a termination factor will be reinvestigated.