Constraints on spacing between transcription factor binding sites in a simple adenovirus promoter

The adenovirus 2 E1B transcription unit has an extremely simple promoter consisting of a TATA box and a closely situated GC box. The interaction between the TATA box and the GC box was analyzed using insertion mutations which expand the distance between the two elements. We observed that the E1B promoter has an unusually rigid architecture. When the GC box, which is a binding site for transcription factor Sp1, was separated further from the TATA box than in the wild-type promoter, in vivo transcription quickly diminished to a level comparable to the elimination of the Sp1 site. Yet all the insertion mutants bound Sp1 factor in vitro with an affinity approximately equal to that of the wild-type promoter. From these results, we argue that Sp1 binding alone is not sufficient to stimulate transcription. The increased distance might disrupt direct contacts between Sp1 and transcription factors bound at the TATA box, contacts required for transcription stimulation by Sp1. The insertion mutations do not interfere with the transcription activation process mediated by the adenovirus large E1A protein or the pseudorabies virus immediate early protein.