The AraC protein, which regulates the L-arabinose operons in Escherichia coi, was dissected into two domains that function in chimeric proteins. One provides a dimerization capability and binds the ligand arabinose, and the other provides a site-specific DNA-binding capability and activates tanscription. In vivo and in vitro experiments showed that a fusion protein consisting of the N-terminal half of the AraC protein and the DNA-binding domain of the LexA repressor dimerizes, binds weDl to a LexA operator, and represses expression ofa LexA operator-3-galactosidase fusion gene in an arabinose-responsive manner. In vivo and in vitro experiments also showed that a fusion protein consisting of the C-terminal half of the AraC protein and the leucine zipper dimerization domain from the C/EBP transcriptional activator binds to aral and activates transcription from a PB.4Dpromoter-,-galactosidase fusion gene. Dimerization was necessary for occupancy and activation of the wild-type AraC binding site.
The genome of the cyanobacterium Synechococcus sp. strain PCC 7942 contains three psbA genes which encode two forms of the Dl protein of photosystem II. Experiments using psbA-lacZ translational fusions and Western blot (immunoblot) analysis have shown that the psbA genes respond differently to changes in light intensity, altering the ratio of the two forms of Dl in the thylakoid membrane. Each gene produces a 1.2-kilobase (kb) mRNA. A probe specific for psbAII transcripts also identified a 1.6-kb mRNA which starts 419 base pairs upstream of the 5' end of the 1.2-kb species and overlaps the entire 1.2-kb transcript. This 419-base-pair region includes an open reading frame (ORF1) of 114 amino acids. We investigated the effects of changes in light intensity on psbAII transcript levels in a series of light shift experiments in the wild-type Synechococcus sp. and in AMC084, a mutant which does not produce the 1.6-kb transcript. After exposure to high light intensities for 15 min, the level of the 1.2-kb psbAII transcript increased in both strains. This transcript was not detected in either strain after transfer to low light intensity. The psbAIII transcript showed the same pattern of response as the 1.2-kb psbAII transcript, whereas the 1.6-kb psbAII transcript was unaffected by different light intensities. The psbAI transcript levels responded oppositely to those ofpsbAII and psbAIII. These data, considered along with previous results obtained by using lacZ translational gene fusions, indicate that the response of psbA genes to changes in light intensity is controlled primarily at the transcriptional level.
The psbDI and psbDII genes in Synechococcus sp. strain PCC 7942 encode the D2 polypeptide, an essential component of the photosystem II reaction center. Previous studies have demonstrated that transcripts from psbDII, but not psbDI, increase in response to high light intensity. Soluble proteins from Synechococcus cells shifted to high light were found to have affinity for DNA sequences upstream from the psbDII coding region. DNA mobility-shift and copper-phenanthroline footprinting assays of a 258-bp fragment revealed three distinct DNA-protein complexes that mapped to the untranslated leader region between +11 and +84. Deletion of the upstream flanking region to -42 had no effect on the expression of a psbDII-lacZ reporter gene or its induction by light, whereas a promoterless construct supported only minimal background levels of I8-galactosidase. A 4-bp deletion within the first protected region of the footprint decreased the 0-galactosid4se activity to approximately 2% of that of the undeleted control, but gene expression remained responsive to light. Deletion of the three protected regions completely abolished both gene expression and light induction. These results suggest that the psbDII gene requires elements within the untranslated leader region for efficient gene expression, one of which may be involved in regulation by light.Cyanobacteria make up a large and diverse group of photosynthetic bacteria. They share with plants the ability to use two photosynthetic reaction centers linked in series, termed photosystems I and II, to reduce photooxidized chlorophyll with electrons obtained from water, releasing molecular oxygen as a by-product. Components of the photosynthetic apparatus of cyanobacteria are highly conserved with their counterparts in the chloroplasts of higher plants (4). The reaction center core of photosystem II contains a dimer of two structurally similar proteins, Dl and D2 (48), which are encoded by the psbA and psbD genes, respectively (4). Dl and D2 house the photoreactive chlorophyll, primary acceptor, and other cofactors involved in electron transport through photosystem II (25,33,48). Both psbA and psbD are unique genes in the chloroplast genomes of most plants (19,36,41,53) but are present in more than one copy in cyanobacterial genomes (9,15,18
A genetic method was developed to determine, in proteins, areas which are tolerant of insertions and deletions. Attractive candidates for these areas are linker regions. Such a region was found to include positions 171 to 178 in the Escherichia coli regulatory protein AraC. Independent biochemical methods identified amino acid residues 11 to 170 as the minimal dimerization domain of AraC, and amino acid residues 178 to 286 out of the 291 residue protein as the minimal DNA-binding domain. Hence, by both the genetic and biochemical approaches, the interdomain linking region was determined to include amino acid residues 171 to 177. The properties of altered proteins were examined using templates with AraC half-sites more widely separated than in the wild-type case. Both AraC protein containing an insertion in the interdomain linker region and a protein consisting of the minimal functional dimerization and DNA-binding domains separated by a 39 amino acid residue linker were able to bind to and function on such a DNA site. In vitro, the proteins with longer linkers bound substantially more stably than wild-type AraC to the DNA containing half-sites for AraC separated by an extra two helical turns of DNA. In vivo on an ara promoter with the more widely separated AraC half-sites, the proteins could activate transcription much better than wild-type AraC.
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