trans activation of the simian virus 40 late promoter by large T antigen requires binding sites for the cellular transcription factor TEF-1

Abstract

Simian virus 40 (SV40) T antigen stimulates the level of transcription from several RNA polymerase II promoters, including the SV40 late promoter. The mechanism of trans activation appears to be indirect since binding of T antigen to specific DNA sequences is not required. However, specific promoter elements that respond to T antigen have not previously been defined. We identified DNA sequences from the SV40 late promoter whose ability to stimulate transcription is induced by the expression of T antigen. In particular, the Sph I + II motifs of the SV40 enhancer can confer T-antigen inducibility to the normally uninducible herpes simplex virus thymidine kinase gene promoter when multiple copies of the sequence are inserted 5' of the transcription initiation site and TATA sequence. Binding sites for the cellular transcription factor TEF-1 and octamer binding proteins are contained within the Sph I + II motifs, as well as at other positions in the SV40 promoter. To study the role of individual protein-binding sites in trans activation by T antigen, mutations were constructed in various TEF-1 and octamer protein-binding sites of the SV40 late promoter. These mutations did not significantly affect basal promoter activity. However, mutation of all three TEF-1 sites prevented detectable activation by T antigen. DNase I footprinting of the mutated promoters with purified proteins demonstrated that inducibility by T antigen correlated with binding affinity of TEF-1 for the DNA and not with binding affinity of an octamer binding protein.

Characterization of the transcription activation function and the DNA binding domain of transcriptional enhancer factor-1.

“…TEF-1 was first identified as a HeLa cell protein that binds cooperatively to tandem repeats of the GT-IIC or Sph enhansons from the simian virus 40 (SV40) enhancer (Davidson et al, 1986(Davidson et al, , 1988Wildeman et al, 1986;Xiao et al, 1987). These TEF-1 binding sites, which have highly degenerate nucleotide sequences, activate transcription from the SV40 early promoter (Davidson et al, 1986;Herr and Clarke, 1986;Zenke et al, 1986;Nomiyama et al, 1987;Ondek et al, 1987Ondek et al, , 1988Schirm et al, 1987;Fromental et al, 1988) and mediate large T antigen activation of the SV40 late promoter (May et al, 1987;Casaz et al, 1991;Gruda and Alwine, 1991;Kelly and Wildeman, 1991). The latter effect possibly involves direct interaction between TEF-l and large T antigen (Gruda et al, 1993).…”

The major transcriptional transactivation domain of simian virus 40 large T antigen associates nonconcurrently with multiple components of the transcriptional preinitiation complex

“…It contains ATPase and helicase activities, binds DNA, directs viral DNA replication, represses transcription from the SV40 early promoter, and binds the tumor suppressor proteins Rb, p53, and p107. Tag is also a promiscuous transactivator of most cellular and viral promoters, including the SV40 late promoter (references 18 and 48 and references cited therein) which can be activated by both TEF-1-dependent (8,32) and TEF-1-independent mechanisms (7). Much of Tag's transactivation of the SV40 late promoter is achieved by inducing replication of the viral genome to a high template copy number, thereby enabling the titration of cellular repressors of this promoter (60).…”

A TEF-1-independent mechanism for activation of the simian virus 40 (SV40) late promoter by mutant SV40 large T antigens

“…CV-1 cells were maintained in Dulbecco's modified Eagle medium (Hazelton) with 10% (vol/vol) newborn calf serum (Gibco), 50 U of penicillin G per ml, and 50 g of streptomycin (Gibco) per ml. Transfections were performed by the calcium phosphate precipitation method (20), modified as described previously (11). Transfection mixtures generally included 2 g of CAT reporter plasmid DNA; 0 or 2 g of either p6-1, to express wild-type T antigen, or plasmids encoding mutant T antigens; 0.5 g of pXGH5 or pTKGH; and pBluescript SKϩ (Stratagene) to bring the total amount of DNA to 20 g/10-cm-diameter plate.…”

“…T antigen activates the late promoter through a replication-independent stimulation of late promoter activity (6,26). Mutagenesis of the late promoter has indicated that binding sites for the cellular transcription factor TEF-1 are necessary for activation by T antigen (11,17,27,28,47). In addition, these sites are sufficient to confer T-antigen inducibility on heterologous promoters (11,21,22).…”

“…Mutagenesis of the late promoter has indicated that binding sites for the cellular transcription factor TEF-1 are necessary for activation by T antigen (11,17,27,28,47). In addition, these sites are sufficient to confer T-antigen inducibility on heterologous promoters (11,21,22). T antigen interacts directly with both TEF-1 and TBP, and induction of the late promoter is abolished with T-antigen fragments that fail to bind either of these factors, with a single exception (22).…”