Activity of simian DNA-binding factors is altered in the presence of simian virus 40 (SV40) early proteins: characterization of factors binding to elements involved in activation of the SV40 late promoter

Simian virus 40 (SV40) large T antigen is a multifunctional protein which plays central roles during both lytic and transforming infections by SV40. It is a potent transcriptional activator and increases expression from the SV40 late promoter and from several cellular promoters. To understand better the transcriptional activation activity of large T antigen, we examined its ability to transactivate a set of simple modular promoters containing one of four upstream activation sequences coupled with one of three different TATA box sequences originally constructed and studied by Taylor and Kingston (Mol. Cell. Biol. 10:165-175, 1990). Large T antigen activated transcription from all of these simple promoters. The identity of the TATA box was a more important determinant of the final level of gene expression than was the identity of the upstream activating sequence element. We also determined the ability of a set of mutant SV40 large T antigens to activate a subset of these promoters. Several mutant SV40 large T antigens which had reduced ability to activate the complex SV40 late and Rous sarcoma virus long terminal repeat promoters showed reduced transcriptional activation activity on all of the modular promoters tested. We used a set of promoter derivatives of the human U6 small nuclear RNA promoter containing different TATA boxes and found that wild-type large T antigen could activate transcription from all of them, although to widely different levels of expression.

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Efficient transcriptional activation of many simple modular promoters by simian virus 40 large T antigen

Rice

Cole

1993

“…Several mutants which transactivated the RSV LTR less well than did wild-type T transactivated the SV40 late promoter at wild-type levels. This probably reflects the fact that these two transcriptional control regions are quite different (21,25,29). Different transcription factors are involved in transcription from these two promoters, and therefore the role played by T antigen in transactivation of each may be different.…”

Section: In303 In346mentioning

confidence: 99%

“…Many studies have focused on defining sequence elements within the SV40 late promoter that are involved in mediating transactivation by T antigen (10,38). Other studies have demonstrated that the presence of T antigen causes alterations in the pattern of cellular transcription factors able to recognize these key sequences, but the mechanism by which these changes are caused is not known (4,20,21).…”

mentioning

confidence: 99%

Mapping the transcriptional transactivation function of simian virus 40 large T antigen

Zhu

Rice

Chamberlain

et al. 1991

T antigen is able to transactivate gene expression from the simian virus 40 (SV40) late promoter and from several other viral and cellular promoters. Neither the mechanisms of transactivation by T antigen nor the regions of T antigen required for this activity have been determined. To address the latter point, we have measured the ability of a set of SV40 large T antigen mutants to stimulate gene expression in CV-1 monkey kidney cells from the SV40 late promoter and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. Transactivation, although reduced, was retained by an N-terminal 138-amino-acid fragment of T antigen. Mutants with alterations at various locations within the N-terminal 85 amino acids transactivated the RSV LTR promoter less well than did wild-type T antigen. Most of these were also partially defective in their ability to transactivate the SV40 late promoter. Two mutants with lesions in the DNA-binding domain that were unable to bind to SV40 DNA were completely defective for transactivation of both promoters, while a third mutant with a lesion in the DNA-binding domain which retained origin-binding activity transactivated both promoters as well as did wild-type T antigen. Only a low level of transactivation was seen with mutant T antigens which had lesions in or near the zinc finger region (amino acids 300 to 350). Mutations which caused defects in ATPase activity, host range/helper function, binding to p53, binding to the retinoblastoma susceptibility protein, or nuclear localization had little or no effect on transactivation. These results suggest that the N-terminal portion of T antigen possesses an activation activity. The data are consistent with the idea that the overall conformation of T antigen is important for transactivation and that mutations in other regions that reduce or eliminate transactivation do so by altering the conformation or orientation of the N-terminal region so that its ability to interact with various targets is diminished or abolished.

“…Both the 72-bp and 21-bp elements can promote transcription in an orientation-independent manner, and factors that bind to them are potential targets for TAg. Recently, sequences within the enhancer have been shown to play a role in TAg activation (5,8,14), and it is hoped that cell-free transcription systems will permit biochemical characterization of the molecules involved.…”

Section: Earlymentioning

confidence: 99%

Activation of simian virus 40 transcription in vitro by T antigen

Coulombe

Berger

Smith

et al. 1992

Simian virus 40 is repressed when the viral early gene product large tumor antigen (TAg) binds to specific sites within the viral origin and DNA replication ensues. Late transcription is activated by TAg, even in the absence of viral DNA replication. We show here that TAg produced in human 293 cells can selectively activate Simian virus 40 transcription in a cell-free system. In the absence of DNA binding by TAg, early and late transcription are both activated, as they are in vivo, suggesting that the effect might be mediated by a cellular component(s) utilized by both the early and late promoters. When TAg binds to the viral origin of replication, early transcription is repressed but the late promoter activation is unaffected. Various preparations of TAg differed in their activities, with some able both to bind DNA and to activate transcription and others able to do only one or the other. Since these variations might be explained by variable amounts of associated factors that copurified with TAg, we asked whether a bacterially derived protein could regulate transcription. An NH2-terminal 272-amino-acid fragment of TAg, produced in Escherichia coli as a glutathione S-transferase fusion protein, retains the ability to activate transcription in vitro, similar to that of the full-length protein. Structural features of this region that might be important are discussed.