Both site-directed and spontaneous mutagenesis have been used to investigate the role of the cis-acting regulatory region between -92 and -1 base pair (bp) of the puc operon of Rhodobacter sphaeroides. The DNA sequence from -84 to -66 bp upstream of the 5` end of the start site of puc operon transcription is essential for normal puc operon expression. This regulatory effect was exerted irrespective of the presence or absence of additional upstream regulatory sequences extending from -629 to -93 bp. It is likely that this region is involved in activator binding. Additionally, two regions of dyad symmetry centered at -42 and -17 bp are shown to be involved in oxygen repression of puc operon expression. Mutations within these regions of dyad symmetry were further subdivided on the basis of whether or not the upstream regulatory region was required to observe the mutant phenotype. Based upon these observations we conclude that these regions of dyad symmetry possessing the motif TGT-N-ACA (where N represents any nucleotide) are involved in repressor binding with the puc operon promoter overlapping each of these dyad symmetries.

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