GENETIC AND BIOCHEMICAL ANALYSIS OF AD5 REGION 2

本提案中所述工作的目标是， 了解控制机制的运作， 真核基因表达，使用腺病毒5型（Ad5）早期区域2A 作为模型系统。 早期区域2A的基因产物是529个氨基酸残基。 酸性单链DNA结合蛋白（DBP），参与病毒 DNA复制，病毒的宿主范围，病毒组装， 早期区域4（E4）的转录和来自早期区域的mRNA的稳定性。 区域1A和1B。 拟议工作的近期目标是在 参与DNA结合和DNA复制的DBP结构域。 三 迄今为止，已经确定了一些区域，其他区域也可能 蛋白质也参与其中。 每个被确定的区域将 进行饱和诱变，以确定 个别氨基酸对DBP在DNA结合和DNA 复制的 将测定突变DBP的能力， 参与腺病毒DNA的起始和延伸反应 体外复制。 DBP突变体将被用于检查蛋白质在控制 转录从E4启动子和稳定的mRNA从E1A和 E1B 将通过北方印迹法测量RNA的稳态水平 分析，以及通过核分析测量的mRNA的合成和衰变速率。 连续和连续标记技术。 DBP在自动调节中的可能作用将通过检查 蛋白质对同源和异源mRNA的亲和力，及其 影响这些RNA的体外翻译。 RNA将被合成 在体外，与E2A和其他mRNA的不同区域的结合通过 过滤器结合试验，以及DBP对体外 这些mRNA的翻译。 DBP和腺病毒DNA聚合酶之间的关联已经被证实。 假设在复制的进动性质中是重要的，但是 这种联系从未得到证实。 我们将使用合成 肽对应的DBP的区域，以确定是否这种潜在的 关联可以被破坏，如果是这样，DBP的哪些区域是 涉案 这些实验的成功结果将为我们提供深入了解 真核基因表达调控的分子机制。

The objective of the work described in this proposal is to gain an understanding of the control mechanisms operative in the regulation of eukaryotic gene expression, using adenovirus type 5 (Ad5) early region 2A as the model system. The gene product of early region 2A is a 529 amino acid single-stranded DNA binding protein (DBP) that is involved in viral DNA replication, the host range of the virus, virus assembly, the transcription of early region 4 (E4) and the stability of mRNAs from early regions 1A and 1B. The immediate goal of the proposed work is to precisely define within the DBP domains that are involved in DNA binding and DNA replication. Three regions have been identified to date, and it is possible that other regions of the protein are also involved. Each region that is identified will be subjected to saturation mutagenesis to define the contribution of individual amino acids to the function of the DBP in DNA binding and DNA replication. The mutant DBPs will be assayed for their ability to participate in the initiation and elongation reactions of adenovirus DNA replication in vitro. DBP mutants will be used to examine the role of the protein in controlling transcription from the E4 promoter and the stability of mRNAs from E1A and E1B. Steady-state levels of RNAs will be measured by Northern blot analysis, and rates of synthesis and decay of mRNAs measured by nuclear run-on and continuous labeling techniques, respectively. A possible role for the DBP in autoregulation will be explored by examining the affinity of the protein for homologous and heterologous mRNAs, and its effect on the in vitro translation of these RNAs. RNAs will be synthesized in vitro, binding to various regions of E2A and other mRNAs measured by filter binding assays, and the effect of the DBP on the in vitro translation of these mRNAs examined. An association between the DBP and the adenovirus DNA polymerase has been postulated to be important in the precessive nature of replication, but such an association has never been demonstrated. We will use synthetic peptides corresponding to regions of the DBP to determine if this potential association can be disrupted, and if so, which regions of the DBP are involved. The successful outcome of these experiments will provide insight into the molecular mechanisms by which eukaryotic gene expression is regulated.