DNA constraints on transcription activation in vitro

Activators of eukaryotic transcription often function over a range of dista nces. It is commonly hypothesized that the intervening DNA between the tran scription 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, activ ation should be sensitive to the presence of intrinsic bends in the interve ning DNA. Similarly, the precise helical phasing of such DNA bends and of t he activator binding sites relative to the basal promoter should affect the degree of transcription activation. To explore these considerations, we de signed transcription templates based on the adenovirus E4 promoter suppleme nted with upstream Gal4 activator binding sites. Surprisingly, we found tha t neither insertion of intrinsically curved DNA sequences between the activ ator binding sites and the basal promoter, nor alteration of the relative h elical alignment of the activator binding sites and the basal promoter sign ificantly affected in vitro transcription activation in HeLa cell nuclear e xtract. In all cases, the degree of transcription activation was a simple i nverse function of the length of intervening DNA. Possible implications of these unexpected results are discussed. (C) 2000 Academic Press.