Mutational effect of the ''-35" element of sorghum psbA gene promoter

Mutations of the first position T and the third position G in TTGACA, the " - 35" element of sorghum psbA gene promoter, were induced using chemically synthesized 20 nt oligonucleotide primer. Three mutants were produced: ATT ACA, GTGACA, and ATGACA. Then the protein binding affinity of the mutants a nd the wild type sorghum psbA gene promoter was tested in a spinach chlorop last protein extract system. Gel retardation assay of the wild type showed a strong protein-binding band. On the other hand, the protein-binding band of the mutant resulting from single base mutation, ATGACA or GTGACA, showed reduced intensity, while that of the mutant resulting from double base mut ation, ATTACA, showed increased intensity. It is thus shown that the " - 35 " element plays an important role in controlling the binding between psbA g ene promoter and the specific chloroplast proteins; mutation of a single ba se may exert a substantial influence on the binding affinity.