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.(ABSTRACT TRUNCATED AT 250 WORDS)