Loewenstein P M, Green M
Institute for Molecular Virology, St. Louis University Medical Center, Missouri 63110.
J Biol Chem. 1989 Dec 25;264(36):21504-8.
The adenovirus E1A gene encodes a protein that transcriptionally activates viral early genes. We have reported that a 49-amino acid chemically synthesized adenovirus type 2 E1A region 3 peptide, PD3 (residues 140-188 of the 289-amino acid protein), can stimulate transcription in vitro from the adenovirus major late promoter. Here we describe reaction properties of E1A trans-activation in vitro with the major late promoter and the early gene 3 promoter and the structural requirements for activity. Stimulation of transcription by PD3 is highest with low levels of DNA template and nuclear extract, and the presence of PD3 eliminates the need to preincubate template with nuclear extract to achieve optimal transcription. These findings suggest that PD3 facilitates a rate-limiting step in the formation of a promoter complex. Analysis of deletion and cysteine substitution mutant PD3 peptides indicates that the C-terminal 70% of the peptide is sufficient for trans-activation in vitro and supports the hypothesis that PD3 contains two functional subregions. The function of one region (residues 140 to about 152) can be overridden under conditions used for in vitro transcription. The second region (residues 153-188) is essential and may function both as a promoter-binding region and as an activating region in vitro.
腺病毒E1A基因编码一种能转录激活病毒早期基因的蛋白质。我们曾报道，一种由49个氨基酸化学合成的2型腺病毒E1A区域3肽，即PD3（289个氨基酸蛋白质的第140 - 188位氨基酸），能在体外刺激腺病毒主要晚期启动子的转录。在此我们描述了E1A体外反式激活主要晚期启动子和早期基因3启动子的反应特性以及活性的结构要求。在低水平DNA模板和核提取物存在时，PD3对转录的刺激作用最强，并且PD3的存在消除了模板与核提取物预孵育以实现最佳转录的必要性。这些发现表明PD3促进了启动子复合物形成过程中的一个限速步骤。对缺失和半胱氨酸替代突变体PD3肽的分析表明，该肽C端的70%足以在体外进行反式激活，并支持了PD3包含两个功能亚区的假说。在用于体外转录的条件下，一个区域（第140至约152位氨基酸）的功能可能会被覆盖。第二个区域（第153 - 188位氨基酸）是必需的，并且在体外可能既作为启动子结合区域又作为激活区域发挥作用。
