| dc.description.abstract | Cyanobacteria perform oxygenic photosynthesis using a two-photosystem photosynthetic apparatus. This apparatus is similar to the thylakoid membrane system of higher plant chloroplasts (1). Photosystem II (PSII) is the complex at which H20 is split, liberating O2, The PSII reaction center consists of the following: 47 and 43 kilodalton (kDa) chlorophyll binding polypeptides, Dl and D2 polypeptides of approximately 32 kDa and cytochrome b-559 (2). Dl is encoded by the psbA gene which is highly conserved among plants and cyanobacteria. Higher plants contain a single copy of the psbA gene in the chloroplast genome (3,4), while cyanobacteria have multigene psbA families (5,6).
Synechococcus strain sp. PCC 7942, a cyanobacterium is useful for studying photosynthesis because it is highly transformable, i. e. it can take up and incorporate exogeneous DNA (8). This is an important feature for using recombinant DNA methods to study gene expression. Synechococcus contains three different copies of psbA, encoding two forms of Dl (7,8). It has been shown that each gene is capable of producing sufficient functional D1 to support photosynthesis in an actively growing culture (8). Form I, the product of psbAI, differs from Form II, the product of psbAll and psbAIII, at 25 of 360 amino acid positions. Twelve of these positions are in the first 16 amino acids of the protein.
Gene fusion experiments (9) have been done to monitor the expression of the psbA genes in Synechococcus at different light intensities. Translational fusions between each of the Synechococcus psbA genes and the Escherichia coli lacZ gene were inserted into the chromosome of wild-type Synechococcus at the respective psbA loci to serve as in vivo reporters of psbA expression. β-Galactosidase (product of lacZ) activities indicated differential expression of the psbA-lacZ gene fusions related to light availability. Expression of psbAI was greater than the expression of psbAII and psbAIII under similar conditions. As light intensity decreased, expression of the psbAl reporter increased, whereas expression of the psbAII and psbAIII reporters decreased. In another study (10), Northern blot analysis was used to measure the levels of the Synechococcus psbA genes in a series of light shift experiments. After 15 minutes of exposure to high light intensities, the levels of a 1.2 kilobase (kb) psbAII transcript and the psbAIII transcript increased, while the psbAl transcript levels responded oppositely. There was also a 1.6 kb psbAII transcript which was unaffected by the changes in light intensity. In each of these studies the expression of the psbA genes was measured by mRNA levels or the levels of a reporter enzyme produced from gene fusions. These data, taken together, indicate that transcriptional regulation plays an important role in the light regulated expression of the psbA genes. This study focuses on the transcriptional regulation of the psbAIII gene in Synechococcus.
There are many examples, in prokaryotes and eukaryotes, of transcriptional regulation by proteins binding upstream of the open reading frame of a gene (11). These regulatory proteins act to activate or repress the transcription of the regulated gene. The binding sites are usually located in the promoter region or further upstream.
DNA mobility shift assays were done to determine if factors in Synechococcus protein extracts bound to the upstream region of psbAIII. The assays were repeated with smaller fragments to define the general binding area. A 190 bp fragment corresponding to a region flanking psbAIII formed DNA-protein complexes with Synechococcus protein(s). This fragment includes the promoter, approximately 50 additional upstream bases, the translational start site, and about 100 bases of the coding region of psbAIII. The binding reaction was optimized, and copper-phenanthroline DNA footprinting was done to identify the bases involved in the binding reaction. | en |
