Organization of the transcriptional control region of the E1b gene of adenovirus type 5.

Genetic analysis of the transcriptional control sequences of the E1b gene of adenovirus type 5 identified two regions that stimulated specific transcription by whole cell extracts from uninfected cells. The first region, located within 50 nucleotides (position -50) 5' to the transcription initiation (cap) site, contains a G+C-rich consensus-binding site (GC box) for the transcription factor Sp1 and a TATA box. Unambiguous stimulatory activity of the second region, between positions -358 and -127, was observed only in the absence of the GC box. DNase I protection experiments (footprinting) with crude nuclear extracts from uninfected cells revealed multiple DNA-protein interactions at the control region. Proximal to the initiation site, both the GC box and the cap site were protected; however, protection of the TATA box was not observed. In the distal region, four protein-binding sites, designated I through IV, were located between positions -250 and -120. Three of the four mapped in protein-coding sequences of the adjacent E1a gene. Sites I and II were 5' to position -218 whereas sites III and IV were 3' to position -218. This finding was consistent with results of the transcriptional analysis indicating that subsets of the distal region were sufficient for stimulation of transcription in vitro in the absence of the GC box. Within the boundaries of site I, a 10-base-pair protected sequence was similar to one located 5' to the adenovirus E1a, E2a, E3, E4, E2 late, and polypeptide IX transcription initiation sites. Sequences within the boundaries of the other three sites were similar to those within other viral and cellular enhancers.