UBC Theses and Dissertations
Studies on the transcription of photosynthesis genes of the photosynthetic bacterium Rhodobacter capsulatus Forrest, Mary Elspet
Rhodobacter capsulatus is a Gram negative bacterium that exhibits a variety of growth modes, including chemoheterotrophic growth and photoheterotrophic growth. Upon a shift of cultures from high to low oxygen concentrations the photosynthetic apparatus is synthesized and incorporated into the inner membrane. The puf operon contains genes that encode structural proteins found in the light-harvesting and reaction center complexes. In a preliminary attempt to pinpoint the location of the puf promoter R. capsulatus RNA polymerase was purified by standard techniques and used in in vitro runoff transcription assays. It was found that the polymerase was capable of specific transcription with linearized pUC13 DNA but no specific transcription could be obtained with K capsulatus DNA. It was concluded that some factor or condition necessary for specific transcription with R capsulatus DNA was absent from these assays. The location of the puf promoter was subsequently found through a series of deletions and oligonucleotide-directed mutations in the 5' region of the puf operon. Fragments that contained these mutations were placed translationally in-frame with the lacZ gene of Escherichia coli in plasmids that could be conjugated into K capsulatus. Assays of beta-galactosidase activities under low and high oxygen conditions resulted in localization of the promoter to a position approximately 540 basepairs upstream of what was previously believed to be the first gene of the operon, the pufB gene. RNA 5' end-mapping experiments showed that the quantity of RNA transcripts obtained were comparable to the lacZ activities. The existence of multiple low abundance RNA 5' ends prompted the theory that the primary transcript has a short half-life, and is rapidly processed to yield a more stable transcript with a 5' end that maps just upstream of the pufB gene. It was found that only the 5' end nearest to the promoter could be capped by guanylyl transferase, and this could only be detected when the putative processing sites were deleted. The DNA sequence between the promoter and the pufB gene contains a new gene of the puf operon, the pufO gene. Deletion of this gene showed that it plays an essential role in the formation of mature light-harvesting and reaction center complexes.
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