Transcription stimulation of the adenovirus type 12 E1a gene in vitro by the 266-amino-acid E1A protein

Abstract

We previously showed that the 13S but not the 12S mRNA product of the Ela gene of the highly oncogenic type 12 adenovirus (Adl2) stimulates the expression of its own gene. In this study, the mechanism for the autoregulation of the Adl2 Ela gene was investigated in vitro. The 266-amino-acid ElA protein of Adl2 was synthesized in yeast cells and purified as a 57-kDa polypeptide. The purified Adl2 ElA 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 EIA protein. The Adl2 ElA protein formed a complex with a TATA box-binding protein (TBP), as does the EIA protein of nononcogenic Ad serotypes. Moreover, the ElA 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 Adl2 Ela gene. These results suggest that the 13S mRNA product of the Adl2 Ela gene regulates the transcription of its own gene by modulating the activity of TBP.

Synergistic activation of NF-?b and inducible isoform of nitric oxide synthase induction by interferon-? and tumor necrosis factor-? in INS-1 cells

“…These represent IFN-␥-activated sites (GAS) in the promoter of the IRF-1 gene, 5Ј-GATCCGATTTCCCCGAAAT-3Ј (Tourkine et al, 1995); GAS in the iNOS gene promoter, 5Ј-CTTTTCCCCTAACAC-3Ј (Gao et al, 1997), and the consensus NF-B binding sequence (B site), 5Ј-GATC-CCAACGGC-AGGGGAATTCCCCTCTCC-3Ј (Khachigian et al, 1995). Electrophoretic mobility shift assay (EMSA) was performed according to a method described elsewhere (Kawamura et al, 1994). The probe (10 4 cpm) was incubated with 10 g of the nuclear protein along with 1 g of poly(dI-dC)⅐poly(dI-dC) (Pharmacia) in each reaction for 30 min at room temperature.…”