Kawamura H, Wada N, Makino Y, Tamura T A, Koikeda S, Shiroki K, Masamune Y, Nakanishi Y
Graduate School of Natural Science and Technology, Kanazawa University, Ishikawa, Japan.
J Virol. 1994 Aug;68(8):5056-62. doi: 10.1128/JVI.68.8.5056-5062.1994.
We previously showed that the 13S but not the 12S mRNA product of the E1a gene of the highly oncogenic type 12 adenovirus (Ad12) stimulates the expression of its own gene. In this study, the mechanism for the autoregulation of the Ad12 E1a gene was investigated in vitro. The 266-amino-acid E1A protein of Ad12 was synthesized in yeast cells and purified as a 57-kDa polypeptide. The purified Ad12 E1A protein stimulated transcription from the proximal promoter of its own gene but had almost no effect on that from the distal promoter. A 35-bp upstream region including a TATA box for the proximal promoter seemed to be sufficient for transcription stimulation by the E1A protein. The Ad12 E1A protein formed a complex with a TATA box-binding protein (TBP), as does the E1A protein of nononcogenic Ad serotypes. Moreover, the E1A protein significantly reduced the binding of TBP to a TATA sequence, while it did not affect the DNA-binding activity of nuclear factor I, a stimulatory protein of the distal transcription of the Ad12 E1a gene. These results suggest that the 13S mRNA product of the Ad12 E1a gene regulates the transcription of its own gene by modulating the activity of TBP.
我们之前发现，高致癌性12型腺病毒（Ad12）E1a基因的13S而非12S mRNA产物可刺激其自身基因的表达。在本研究中，我们在体外研究了Ad12 E1a基因的自调控机制。Ad12的266个氨基酸的E1A蛋白在酵母细胞中合成，并作为57 kDa的多肽进行纯化。纯化的Ad12 E1A蛋白可刺激其自身基因近端启动子的转录，但对远端启动子的转录几乎没有影响。包括近端启动子TATA框在内的35 bp上游区域似乎足以被E1A蛋白刺激转录。与非致癌性Ad血清型的E1A蛋白一样，Ad12 E1A蛋白与TATA框结合蛋白（TBP）形成复合物。此外，E1A蛋白显著降低了TBP与TATA序列的结合，而不影响Ad12 E1a基因远端转录的刺激蛋白核因子I的DNA结合活性。这些结果表明，Ad12 E1a基因的13S mRNA产物通过调节TBP的活性来调控其自身基因的转录。
