TRANSCRIPTION STIMULATION BY ADENOVIRUS E1A PROTEIN

腺病毒E1A蛋白直接参与肿瘤的发生， 通过腺病毒转化。 它是一种核蛋白， 结构和功能同源性的重要产品 myc人癌基因。 E1A蛋白刺激基因转录 通过转染或病毒感染引入哺乳动物细胞。 这个过程的机制，称为E1A反式激活，不是 却又被人理解。 第一个供资期的结果 表明特异性宿主细胞转录因子包括 TFIIIC 1、TFIIIC 2（均在第一个供资期确定）， 而TATA盒转录因子的活性在 E1A的表达。 然而，这些宿主细胞转录 这些因素还没有被纯化到同质化。 从而 无法直接确定E1A的影响 蛋白质的数量和具体活动，这些 转录因子 为了做到这一点，为了追求生物化学， 这些转录因子的活性 增加了E1A蛋白，我们建议纯化这些 转录因子的同质性。 这将使直接 包含这些多肽的多肽的表征 活动 将针对纯化的 蛋白质和用于确定数量和物理性质 的蛋白质在腺病毒和模拟感染的HeLa细胞。 纯化的转录因子也将被用于研究 转录因子的功能 转录起始 TFIIIC 2和TFIIIC 1与 将分析野生型和突变型VAI、tRNA和5S rRNA基因 通过核酸酶保护和结合干扰研究。 的 TATA盒因子与野生型和突变型Ad2的相互作用 E1B启动子已经被很好地表征了其 将分析体内转录。 cDNA和基因编码 将分离TFIIIC 1、TFIIIC 2和TATA盒TF多肽 和分析 这将产生关于氨基酸的信息 这些蛋白质的序列将在它们的 在感染和未感染的细胞中进行表征。 比如说， 这些蛋白质可能是受后 翻译修饰如磷酸化。 氨基酸 序列对于确定修饰位点至关重要。 基因组和cDNA克隆也将有助于分析 这些基因响应E1A的转录调节， 生长因子 拟议研究的结果应 有助于理解转录因子的功能， E1A反式激活的调控机制。

Adenovirus E1A protein is directly involved in oncogenic transformation by adenovirus. It is a nuclear protein with structural and functional homology to the product of the important myc human oncogene. E1A protein stimulates transcription of genes introduced into mammalian cells by transfection or viral infection. The mechanism of this process, called E1A transactivation, is not yet well understood. Results from the first funding period indicate that specific host cell transcription factors including TFIIIC1, TFIIIC2 (both identified during the first funding period), and the TATA-box transcription factor increase in activity after expression of E1A. However, none of these host cell transcription factors have yet been purified to homogeneity. Consequently, it has not been possible to directly determine the effect of E1A protein on the quantity and specific activities of these transcription factors. To do this, and to pursue the biochemical mechanisms by which the activities of these transcription factors are increase by E1A protein, we propose to purify these transcription factors to homogeneity. This will enable a direct characterization of the polypeptides which comprise these activities. Specific antisera will be raised against the purified proteins and used to determine the amounts and physical properties of the proteins in adenovirus and mock infected HeLa cells. Purified transcription factors will also be used in studies of the functions of the transcription factors in transcription factors in transcription initiation. Interactions of TFIIIC2 and TFIIIC1 with wild-type and mutant VAI, tRNA, and 5S rRNA genes will be analyzed by nuclease protection and binding interference studies. The interaction of the TATA-box factor with wild-type and mutant Ad2 E1B promoters which have been well characterized for their transcription in vivo will be analyzed. cDNAs and genes encoding the TFIIIC1, TFIIIC2 and TATA-box TF polypeptides will be isolated and analyzed. This will yield information on the amino acid sequence of the proteins which will be useful in their characterization in infected and uninfected cells. For example, it is possible that these proteins are regulated by post- translational modifications such as phosphorylation. Amino acid sequence would be crucial to defining the sites of modification. Genomic and cDNA clones will also be useful in analyzing the transcriptional regulation of these genes in response to E1A and growth factors. Results form the proposed studies should contribute to an understanding of transcription factor function and regulation and the mechanism of E1A transactivation.