FIG. 3.

DCE subelement III can function independently of subelements I and II. A. HSV U_L38 wild-type (WT) and mutant promoter sequences. Predicted S_III sequences are in blue, and mutated bases are in red (26). In vitro transcriptions of the U_L38 WT promoter and DAS mutants 1 and 3 (das1 and das3) were performed using HeLa nuclear extracts (NE) or a highly purified transcription system containing either TBP or affinity-purified TFIID. Below the in vitro transcription are quantifications of two sets of the in vitro transcription experiments. B. Adenovirus promoter sequences containing DCE sequences. Shown are several promoters from adenovirus 5 (GenBank accession numbers BK000408 and X02996). The promoters are indicated on the left. The arrow indicates the +1 transcriptional start site. The vertical boxes indicate positions that fit the Inr consensus sequence (YYANA/TYY [73]). The three boxes and blue lettering indicate, in the case of the MLP, experimentally derived positions of the three DCE subelements (Fig. 1). In the case of the remaining promoters, the boxes and blue lettering indicate predicted DCE subelements. C. In vitro transcription analysis of the wild-type adenovirus E3 promoter and a mutation of the predicted S_III (E3 mAGC; AGC is mutated to TTT). Shown are transcriptions using HeLa nuclear extracts (NE) or using the highly purified reconstituted transcription system (RTS) containing either recombinant TBP or affinity-purified TFIID. D. Titration of the adenovirus IX promoter containing either the wild-type promoter sequences (IX) or a mutation of the predicted S_III AGC sequence (IX mAGC; AGC is mutated to TTT). Shown are transcriptions using HeLa nuclear extracts. E. DCE subelement III function has severe spatial constraints. Wild-type and mutant E3 in vitro transcriptions using HeLa nuclear extracts are compared to the deletion or insertion of 3 base pairs between the subelement III AGC and the start site of transcription (−3AGC and +3AGC, respectively; the insertion of AAA was at +24 for +3AGC, and deletion of the TCG sequence was at +21 to +23 for −3AGC). F. Analysis of Ad MLP spacing mutants. The Ad MLP S_III was moved 4 bp closer to S_II to establish a 10-bp interval between S_II and S_III as in the β-globin promoter (−4S_III; the GGCC from +28 to +31 was deleted). In this context, S_III was also mutated to TTT (−4mS_III). Shown are results of duplicate in vitro transcriptions (using HeLa NE) of these two templates, compared to the wild-type Ad MLP (MLP).