CONTROL OF ADENOVIRUS TRANSCRIPTION

参与转化的致癌细胞基因的激活， 至少在某些情况下，许多其他基因的表达水平似乎是 转录。 本建议的中心是分析 通过RNA聚合酶II和III的体外系统进行转录， 忠实地再现体内启动的部位和要求， 转录。 我们将使用一种简单的生化 分离方法与遗传方法，其利用 模板和转录机制突变体以及核苷酸 类似物 RNA聚合酶II的工作将集中在蛋白质参与， 通过详细分析生化（酶与辅因子）启动 以及遗传的（即，模板序列中）所需的功能 转录起始复合物的形成。 我们将使用 HeLa细胞的转录提取物和模板， 插入细菌质粒或质粒中的致癌腺病毒DNA。 小说 起始过程的特征，例如，DNA的解旋 双螺旋或转录抑制剂的作用机制 启动，DRB，将是一些部分过程， 在转录起始复合物中进行了详细分析。 退绕 检测试剂盒也将用于RNA聚合酶转录的VAI RNA基因 三. 转录复合体的形成， 在转录的起始和延伸阶段之间，揭示了 引发事件的大摩尔过量。 这种过度可能与 通过延长量的增加而激活基因的机制 因素或可能是体外系统的人工制品。 点突变体 通过对M-13进行位点特异性诱变产生DNA模板 允许突变体的快速序列分析的克隆载体， 用重组DNA的复制形式进行体外转录 噬菌体。 位点特异性点突变体允许更好地建立 启动子序列的每个元件，因为启动子周围的环境 突变保持不变。 突变体或 野生型启动子序列和转录调控元件 机械，细胞RNA聚合酶II α-鹅膏蕈碱抗性突变体和 将分析转录抗性DRB抗性突变体。 RNA聚合酶或因子的突变会产生一些异常的 交互. 我们将继续努力建立这一机制 转录起始抑制剂DRB和生物化学物质的作用 这些细胞突变体的基础。

The activation of oncogenic cellular genes involved in transformation and of many other genes seems to be, at least in some cases, at the level of transcription. This proposal centers on the analysis of initiation of transcription by RNA polymerases II and III in vitro systems which faithfully reproduce the site and requirements for in vivo initiation of transcription. We will use a combination of straight biochemical fractionation approaches with a genetic approach which takes advantage of template and transcriptional machinery mutants as well as nucleotide analogs. The RNA polymerase II work will focus on the proteins involved in initiation by analyzing in detail the biochemical (enzyme with cofactors) as well as genetic (i.e., in the template sequence) features required for the formation of the transcription initiation complex. We will use transcriptional extracts from HeLa cells and templates consisting of oncogenic adenovirus DNA inserted in bacterial plasmids or phages. Novel features of the initiation process, like for example, unwinding of the DNA double helix or the mechanism of action of the inhibitor of transcription initiation, DRB, will be some of the partial processes that will be analyzed in detail in transcriptional initiation complexes. The unwinding assay will also be used for the VAI RNA genes transcribed by RNA polymerase III. The formation of a transcriptional complex, which distinguishes between the initiation and elongation phases of transcription, revealed a large molar excess of initiation events. This excess could be related to a mechanism of gene activation by an increase in the amounts of elongation factors or might be an artifact of the in vitro system. Point mutants in the DNA template will be generated by site-specific mutagenesis on M-13 cloning vectors which allow rapid sequence analysis of the mutants and in vitro transcription with the replicative form of the recombinant DNA phage. Site-specific point mutants allow to better establish the role of each element of the promoter sequence, since the environment around the mutation remains constant. The interactions between the mutant or wild-type promoter sequences and the elements of the transcriptional machinery, cellular RNA polymerase II Alpha-amanitin resistant mutants and transcriptionally resistant DRB resistant mutants will be analyzed. Mutations in the RNA polymerase or factors should produce some abnormal interactions. We will continue our efforts in establishing the mechanism of action of the transcription initiation inhibitor DRB and the biochemical basis for these cell mutants.