Identification of a new cis-regulatory element in a Nicotiana tabacum polyubiquitin gene promoter

The polyubiquitin gene Ubi.U4 is expressed in a complex pattern during cell division and plant development in Nicotiana tabacum. Plants transformed with the uidA reporter gene placed under the control of the proximal 263 bp of the promoter are able to express GUS activity in dividing cells. This expression is modified when a G-box-like motif is mutated. In order to clarify the transcriptional regulation of this gene, we analysed positively regulating sequences in this proximal promoter by electrophoretic mobility-shift assays (EMSA). We observed that the G-box-like element gave rise to only weak protein-DNA bands but that a new motif, GCTGTAC, directs formation of numerous, distinctive, strong 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, present 12 nucleotides upstream of the G-box-like element, thus seems also to be critical for Ubi.U4 promoter activity.