Sequence and Spacing of TATA Box Elements Are Critical for Accurate Initiation from the -Phaseolin Promoter*

The -phaseolin (phas) gene, which encodes one of the major seed storage proteins of P. vulgaris, is tightly regulated at the transcription level resulting in strict tissuespecific and spatial expression during embryonic development. The phas proximal promoter contains a complex arrangement of core promoter elements including three TATA boxes as well as several putative initiator elements. To delineate the respective contributions of the core promoter elements to transcription initiation we have performed site-directed mutagenesis of the phas promoter. In vivo expression studies were performed on transgenic Arabidopsis harboring phas promoter mutants driving expression of the -glucuronidase (gus) reporter gene. Quantitative assessment of GUS activity in seeds bearing the promoter mutants indicated that both sequence and spacing of the TATA elements influenced the efficiency of transcription. Substitution, insertion or deletion mutations had no effect on histochemical staining patterns indicating that strict spacing requirements are not essential for correct spatial expression of phas during embryogenesis. Further evaluation of the phas promoter by in vitro transcription analysis revealed the presence of multiple TATA-dependent transcription initiation start sites. The distance between TATA elements and transcription start sites was maintained in insertion and deletion mutants through the creation of novel initiation sites, indicating that positioning of the TATA elements rather than DNA sequence was the primary determinant of start site location. We conclude that, while dispensable for proper spatial distribution, the complex architecture of the phas promoter is required to ensure high levels of accurate phas transcription initiation in the developing embryo.