Nopaline synthase (nos) is of bacterial origin and expressed in plant tissues to generate an unusual compound, nopaline, which is secreted into the environment where Agrobacterium tumefaciens uses it as a nutrient source. The nos promoter contains three distinctive regions which are important for the promoter activity. Among these, the upstream region between -131 and -112 is essential for the nos promoter. The upstream region consists of two hexamer motifs which are separated by an eight nucleotide sequence. In this study, we have investigated the role of the hexamer motif that is located between -117 and -112. Increasing the distance between the hexamer and the downstream regulatory region significantly reduced the promoter activity, indicating that a proper distance between the regulatory elements is needed for an optimum level of activity. Insertion of a single copy of the hexamer enhanced the promoter in both orientations. Point mutations in the inserted hexamer sequence reduced the enhancing effect. These results confirmed that the hexamer motif is important for the nos promoter activity. The b sequence that is located immediately downstream of the hexamer motif did not function by itself, but together with the hexamer it appeared to enhance the promoter activity. These indicate that the sequence surrounding of the upstream regulatory element is also involved in controlling the promoter function.
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