In the simple adenovirus 2 early 1B (E1B) promoter, moving the single Sp1 transcription factor binding site (GC box) from its wild-type position eight base pairs (bp) from the TATA box to 30 bp upstream is equivalent to its deletion (Wu, L., and Berk, A. (1988) Genes & Dev. 2, 403-411). In more complex promoters, multiple upstream elements regulate transcription over greater distances. To understand these spacing constraints, we placed two GC boxes in phase at various distances from the E1B

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... box. Whereas one GC box at eight bp from TATA increases transcription in vivo 5-fold compared with TATA alone (Wu, L., Rosser, D. S. E., Schmidt, M. C., and Berk, A. (1987) Nature 326, 512-515), two GC boxes increased in vivo transcription 25-30-fold. Transcriptional stimulation by two GC boxes fell off rapidly in vivo at 30 bp from TATA, remained approximately constant through 50 bp, then decreased again at 70 bp. Consequently, two GC boxes have a multiplicative effect at eight bp from TATA and continue to stimulate transcription at a greater distance from TATA than a single Sp1 site. Quantitatively different results were observed for in vitro transcription using a nuclear extract; a more linear fall off with increasing distance from TATA was observed. Separating the two GC boxes progressively decreased transcription. E1A stimulation of these promoters in vivo indicates that Sp1 transcription control is independent of E1A transactivation.