Analysis of the Rhodobacter capsulatus puf operon. Location of the oxygen-regulated promoter region and the identification of an additional puf-encoded gene

Abstract

In an attempt to identify features of an oxygen-regulated promoter, we have determined the location of transcription initiation for the puf operon. The position for the oxygen-regulated promoter was demonstrated by several independent means to be located 699 base pairs (bp) upstream from the pufB structural gene. DNA sequence analysis of the promoter region demonstrates the presence of a 26-base pair region of dyad symmetry followed by a sequence containing homology to promoters which use the RNA polymerase sigma 60 subunit (ntrA) for recognition of DNA. In addition to the oxygen-regulated promoter, a region responsible for low-level constitutive expression of the puf operon was shown to initiate transcription 511 bp upstream from the pufB gene. In contrast to the oxygen-regulated promoter, this second promoter contains no obvious secondary structure nor sequence homology to ntrA-dependent promoters. DNA sequence analysis demonstrates the existence of an additional open reading frame (designated as pufQ) that is located between the promoters and the pufB structural gene. A translational fusion of pufQ to lacZ was used to demonstrate that pufQ is efficiently translated and regulated in a manner analogous to a translational fusion of pufM to lacZ. Finally, we also demonstrate that puf operon transcription initiation and regulation does not involve any puf-encoded gene products.