Transcriptional activation by simian virus 40 large T antigen: interactions with multiple components of the transcription complex
Gruda, M.C.; Zabolotny, J.M.; Xiao, J.H.; Davidson, I.; Alwine, J.C.
Molecular and Cellular Biology 13(2): 961-969
Simian virus 40 (SV40) large T antigen is a potent transcriptional activator of both viral and cellular promoters. Within the SV40 late promoter, a specific upstream element necessary for T-antigen transcriptional activation is the binding site for transcription-enhancing factor 1 (TEF-1). The promoter structure necessary for T-antigen-mediated transcriptional activation appears to be simple. For example, a promoter consisting of upstream TEF-1 binding sites (or other factor-binding sites) and a downstream TATA or initiator element is efficiently activated. It has been demonstrated that transcriptional activation by T antigen does not require direct binding to the DNA; thus, the most direct effect that T antigen could have on these simple promoters would be through protein-protein interactions with either upstream-bound transcription factors, the basal transcription complex, or both. To determine whether such interactions occur, full-length T antigen or segments of it was fused to the glutathione-binding site (GST fusions) or to the Gal4 DNA-binding domain (amino acids 1 to 147) (Gal4 fusions). With the GST fusions, it was found that TEF-1 and the TATA-binding protein (TBP) bound different regions of T antigen. A GST fusion containing amino acids 5 to 172 (region T1) efficiently bound TBP. TEF-1 bound neither region T1 nor a region between amino acids 168 and 373 (region T2); however, it bound efficiently to the combined region (T5) containing amino acids 5 to 383. The Gal4 fusions demonstrated that no region of T antigen could activate a promoter containing Gal4-binding sites, suggesting that T antigen does not contain an activation domain of the type defined by this assay. However, the Gal4 fusion proteins maintained their ability to activate promoters known to be activated by wild-type T antigen. The fusion with region T1, which binds only TBP, modestly activated the SV40 late promoter and the simple TEF-1/TATA promoter. Region T5, which binds both TBP and TEF-1, activated each of these promoters to levels equivalent to that of wild-type T antigen. The correlation between the binding of both TEF-1 and TBP and the ability to mediate wild-type level of transcriptional activation suggests that T antigen causes activation through direct interactions with multiple factors in the transcription complex.