UBC Theses and Dissertations
Studies on the transcription of three overlapping operons encoding photosynthesis genes from the phototrophic bacterium Rhodobacter capsulatus Wellington, Cheryl Lea
Rhodobacter capsulatus photosynthesis gene was isolated by creating in-frame fusions in a lacZ transcriptional/translational vector, and selecting for those that directed oxygen-regulated levels of β-galactosidase in R. capsulatus. One lacZ fusion isolate was used to identify an open reading frame (ORF) of unknown function and flanking sequences that promoted initiation of transcription. Interposon mutagenesis experiments identified the ORF as the bchC gene, which encodes an enzyme that catalyses the penultimate step in the biosynthesis of bacteriochlorophyll a, and also showed that the bchC gene formed an operon with the bchA gene. The nucleotide sequence of this bchC gene and its 5' regulatory region were determined. The deduced amino acid sequence showed that the bchC gene encodes a 33 kDA protein that has hydrophobic segments that could interact with a lipid membrane, and that this putative BchC protein contains a potential bacteriochlorophyll a binding site. Deletion analysis, S1-nuclease protection, and primer extension experiments showed that promoter activity was associated with sequences to which a 5' end mapped, and that these sequences had significant similarity to the proposed promoter regions of several other R. capsulatus photosynthesis genes. RNA blotting and S1-nuclease protection end-mapping experiments using bipartite probes provided direct evidence that the mRNA transcripts of the bchCA operon overlap those of the two flanking operons, the crtEF and the puf operons, such that the crtEF, bchCA, and puf operons may be cotranscribable, and that RNA polymerase may initiate transcription at one of several promoters. The significance of these overlapping mRNAs was evaluated using two interposon mutant strains, one that prevented crtEF transcripts from overlapping those the bchCA and puf operons, and the other that prevented both crtEF and bchCA transcripts from overlapping those of the puf operon. The results suggested that transcriptional readthrough stimulates promoter activity. Moreover, a pufB::lac'Z fusion could be expressed from the bchCA promoter equally as well as from the puf promoter, suggesting that these overlapping transcripts are functionally significant in the chromosomal context.
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