LOCALIZATION OF THE 12-O-TETRADECANOYLPHORBOL-13-ACETATE RESPONSE OF THE HUMAN ORNITHINE DECARBOXYLASE PROMOTER TO THE TATA BOX

In a previous study, we narrowed the region of the human ornithine dec arboxylase (ODC) promoter responsive to 12-O-tetradecanoylphorbol-13-a cetate (TPA) to nt -42 to +54 around the transcription initiation site (Kim YJ, Pan H, Verma AK, Mol Carcinog 10:169-179, 1994). Here we rep ort defining the role of the TATA box in TPA-induced transcription fro m the -42/+54 ODC promoter fragment. A transversion mutation at the th ird position of the TATA box (<TA(T)under bar AAGT>--><TA(A)under bar AAGT>) reduced TPA responsiveness of the reporter construct -42/+54 OD C-Luc by 49%. Electrophoretic mobility shift assays (EMSAs) using HeLa cell nuclear protein extracts revealed no differences in the binding pattern between the natural -42/+54 ODC promoter element and the -42/54 ODC promoter element containing the T-->A mutation. However, antibo dies to the general transcription factor TFIIB disrupted the DNA-prote in complexes normally formed with the -42/+54 ODC promoter element in EMSAs. A consensus TATA box oligonucleotide formed two bands, with the faster mobility band displaying enhanced binding with nuclear protein extracts from TPA treated cells. Furthermore, incubation of HeLa cell nuclear extracts with an oligonucleotide containing the ODC TATA box also caused formation of two specific bands in EMSA. Both bands exhibi ted augmented binding to nuclear proteins from TPA-treated cells. Intr oduction of the T-->A transversion mutation in the ODC TATA oligonucle otide eliminated binding of the faster migrating band formed with the natural ODC TATA oligonucleotide. These results indicate that TPA modu lation of the general transcription machinery may play a role in the T PA-activated transcription of the human ODC promoter. (C) 1996 Wiley-L iss, Inc.