SV40 large tumor antigen (T antigen) is a potent viral oncoprotein which is capable of a large array of biochemical functions. These functions affect DNA replication, cell cycle control and the control of transcription. Much of our previous work has dealt with the transcriptional activation function of T antigen using the SV40 late promoter as a model system. However, T antigen is a promiscuous activator of many heterologous promoters. This may account, at least in part, for the dramatic alterations in cellular gene expression seen during transformation and lytic infection. Our analyses of the transcriptional activation mechanisms mediated by T antigen have shown that the promoter structure necessary for activation is relatively simple: a TATA or initiator element with an upstream binding site for a transcription factor. There is a great deal of variability allowed in these promoters, which may explain T antigen's promiscuous activation. Our data suggest that the late promoter is made up of a number of these more simple promoters. Using simple promoter constructions we have found that the overlapping binding sites for octamer and TEF transcription factors, form a specific upstream region through which T antigen mediates transcriptional control of the late promoter. Activation is mediated through TEF, while octamer factor binding mediates negative regulation of late promoter activity. Evidence suggests that octamer and/or TEF may be modified in T antigen containing cells. However, the number of different promoters activated by T antigen indicates that it may also affect many other factors. The utilization of synthetic, simple promoters allows us to determine promoter structures required for activation by T antigen. Therefore, in Aim 1 we propose to utilize well defined promoters to identify: 1) the simplest promoter necessary for activation by T antigen; 2) the requirements for most efficient activation, e.g. the factors, the spacing requirements, preferences between TATA and initiator elements; 3) the variability in the promoter elements that can be used; 4) the effects of multiplicity of homologous and heterologous elements, i.e. systematically building more complex promoters. The findings of these studies provide us with different defined promoter elements and structures which we are utilizing, in Aim 2, for the determination of the mechanism by which T antigen activates transcription. With limited and well defined elements in the promoters we have specific factors to examine in order to determine, for example, whether factors are modified or induced, whether T antigen binds them or whether a cellular protein they interact with has been affected by T antigen. The SV40 late leader region has also been implicated in late promoter function and RNA stability. Therefore, in Aim 3, we will include this region in our studies, examining it for elements involved in transcriptional control and RNA stability.
In Aim 4, we propose a complementary approach to our studies of T antigen transcriptional activation. We have expressed T antigen in Saccharomyces cerevisiae and have found that it mediates transcriptional activation an affects cell cycle control. Therefore, we will utilize the genetics of yeast as an alternate system to determine the mechanisms by which T antigen affects transcriptional control and the cell cycle.
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