Ström A C, Ohlsson P, Akusjärvi G
Department of Medical Biochemistry and Microbiology, BMC, Uppsala University, 751 23 Uppsala, Sweden.
J Virol. 1998 Jul;72(7):5978-83. doi: 10.1128/JVI.72.7.5978-5983.1998.
We have previously shown that the nonconserved carboxy-terminal exon of the adenovirus type 2 E1A-289R protein contains two interchangeable sequence elements, auxiliary region (AR) 1 and AR2, that are required for efficient CR3-mediated transcriptional activation of the viral E4 promoter (M. Bondesson, C. Svensson, S. Linder, and G. Akusjärvi, EMBO J. 11:3347-3354, 1992). Here we show that CR3-mediated transactivation of all adenovirus early promoters and the HSP70 promoter requires the AR1 element. We further show that AR2 can substitute for AR1 only when artificially juxtaposed to CR3. AR1 consists of six tandem glutamic acid-proline (EP) repeats and is positioned immediately downstream of CR3. Genetic dissection of AR1 showed that the number of EP repeats in AR1 is critical for CR3 function. Thus, reducing or increasing the number of EP repeats reduces the CR3 transactivation capacity. Furthermore, the introduction of amino acid substitutions into AR1 suggested that the net negative charge in AR1 is of critical importance for its function as an enhancer of CR3-mediated transcriptional activation. Using an in vitro binding approach, we showed that the AR1 element is not part of the CR3 promoter localization signal mediating contact with the Sp1, ATF-2, or c-Jun upstream-binding transcription factors. Previous studies have suggested that the 49-amino-acid sequence constituting CR3 represents the minimal domain required for E1A-induced activation of viral early promoters. Since AR1 was required for efficient CR3-mediated transcriptional activation of all tested promoters, we suggest that the carboxy-terminal boundary for the CR3 transactivation domain should be extended to include the AR1 element.
我们先前已表明，腺病毒2型E1A - 289R蛋白的非保守羧基末端外显子包含两个可互换的序列元件，即辅助区域（AR）1和AR2，它们是病毒E4启动子高效CR3介导的转录激活所必需的（M. Bondesson、C. Svensson、S. Linder和G. Akusjärvi，《欧洲分子生物学组织杂志》11:3347 - 3354，1992年）。在此我们表明，CR3介导的所有腺病毒早期启动子和HSP70启动子的反式激活需要AR1元件。我们进一步表明，只有当AR2人工并列于CR3时，它才能替代AR1。AR1由六个串联的谷氨酸 - 脯氨酸（EP）重复序列组成，位于CR3的紧邻下游。对AR1的基因剖析表明，AR1中EP重复序列的数量对CR3功能至关重要。因此，减少或增加EP重复序列的数量会降低CR3的反式激活能力。此外，在AR1中引入氨基酸替代表明，AR1中的净负电荷对其作为CR3介导的转录激活增强子的功能至关重要。使用体外结合方法，我们表明AR1元件不是介导与Sp1、ATF - 2或c - Jun上游结合转录因子接触的CR3启动子定位信号的一部分。先前的研究表明，构成CR3的49个氨基酸序列代表E1A诱导病毒早期启动子激活所需的最小结构域。由于AR1是所有测试启动子高效CR3介导的转录激活所必需的，我们建议将CR3反式激活结构域的羧基末端边界扩展至包括AR1元件。
