DNA constraints on transcription activation in vitro

Activators of eukaryotic transcription often function over a range of distances. It is commonly hypothesized that the intervening DNA between the transcription start site and the activator binding sites forms a loop in order to allow the activators to interact with the basal transcription apparatus, either directly or through mediators. If this hypothesis is correct, activation should be sensitive to the presence of intrinsic bends in the intervening DNA. Similarly, the precise helical phasing of such DNA bends and of the activator binding sites relative to the basal promoter should affect the degree of transcription activation. To explore these considerations, we designed transcription templates based on the adenovirus E4 promoter supplemented with upstream Gal4 activator binding sites. Surprisingly, we found that neither insertion of intrinsically curved DNA sequences between the activator binding sites and the basal promoter, nor alteration of the relative helical alignment of the activator binding sites and the basal promoter significantly affected in vitro transcription activation in HeLa cell nuclear extract. In all cases, the degree of transcription activation was a simple inverse function of the length of intervening DNA. Possible implications of these unexpected results are discussed.