IN-VIVO AND IN-VITRO CHARACTERIZATION OF THE LIGHT-REGULATED CPCB2A2 PROMOTER OF FREMYELLA DIPLOSIPHON

When exposed to different spectral qualities of light, many cyanobacte ria dramatically alter their phycobilisome rod composition in a proces s termed complementary chromatic adaptation. In the cyanobacterium Fre myella diplosiphon, this response is associated with differential expr ession of the cpcB2A2, cpeBA, and cpeCDE operons, which code for the p hycobiliproteins phycocyanin and phycoerythrin and the phycoerythrin l inker polypeptides, respectively. To define components of the signal t ransduction pathway involved in light-regulated expression of genes en coding phycobilisome polypeptides, we have used in vivo and in vitro t echniques to identify cis-acting sequences and trans-acting factors ne cessary for the regulation of the red-light-inducible cpcB2A2 operon. Deletion of the cpcB2A2 upstream sequences to -76 bp with respect to t he transcription start site had no effect on red-light induction of a cpcB2A2-beta-glucuronidase (GUS) chimeric gene, while deletion to -37 bp abolished GUS expression. Furthermore, a fragment of the cpcB2A2 ge ne from -76 to +25 bp linked to the untranslated leader of cpcB1A1 (a constitutively expressed operon encoding phycocyanin) is sufficient to drive high-level GUS expression in red light. Therefore, the sequence between positions -76 and -37 is necessary for the expression of cpcB 2A2, and the region extending from -76 to +25 is sufficient for red-li ght induction of the operon. Attempts were made to correlate the in vi vo data with protein binding in the region upstream of the transcripti on start site of cpcB2A2. Using in vitro analysis, we detected two pro tein-binding sites in the cpcB2A2 promoter which were localized to pos itions -162 to -122 and -37 to +25. Proteins from both red- and green- light-grown cells interacted with the former site, while only proteins present in extracts from red-light-grown cells interacted with the la tter site. The data from both the in vivo and in vitro analyses sugges t that while two regions upstream of the cpcB2A2 transcription initiat ion site specifically bind proteins, only the binding site bordering t he transcription start site is important for complementary chromatic a daptation.