IDENTIFICATION OF A NEW CIS-REGULATORY ELEMENT IN A NICOTIANA-TABACUMPOLYUBIQUITIN GENE PROMOTER

The polyubiquitin gene Ubi. U4 is expressed in a complex pattern durin g cell division and plant development in Nicotiana tabacum. Plants tra nsformed with the uidA reporter gene placed under the control of the p roximal 263 bp of the promoter are able to express GUS activity in div iding cells. This expression is modified when a G-box-like motif is mu tated. In order to clarify the transcriptional regulation of this gene , we analysed positively regulating sequences in this proximal promote r by electrophoretic mobility-shift assays (EMSA). We observed that th e G-box-like element gave rise to only weak protein-DNA bands but that a new motif, GCTGTAC, directs formation of numerous, distinctive, str ong and developmentally specific shifts with nuclear extracts prepared from various different developmental stages and from dividing cells. Point mutations within this motif abolish formation of the protein-DNA complexes and reduce promoter activity in transient expression assays . This novel putative cis-acting element, which we called the U-box, p resent 12 nucleotides upstream of the G-box-like element, thus seems a lso to be critical for Ubi. U4 promoter activity.