CO2 response element and corresponding trans-acting factor of the promoter for ribulose-1,5-bisphosphate carboxylase/oxygenase genes in Synechococcus sp. PCC7002 found by an improved electrophoretic mobility shift assay
2002
Onizuka, T. (Toray Industries Inc., Kamakura, Kanagawa (Japan). Basic Research Labs.) | Akiyama, H. | Endo, S. | Kanai, S. | Hirano, M. | Tanaka, S. | Miyasaka, H.
We analyzed the promoter of the genes encoding the ribulose-l, 5-bisphosphate carboxylase/oxygenase (rbc) in the cyanobacterium Synechococcus sp. PCC7002 and localized the CO2-regulatory element. Cyanobacterial transformants were constructed with several DNA segments of the rbc promoter fused to the chloramphenicol acetyltransferase (CAT) gene, and their acetyltransferase activities were analyzed under 0.03% and 1% CO2 conditions. We found that the AT-rich element localized from -262 to -291 relative to the rbc translation-starting site was required for CO2-dependent repression. Fluorescent-labeled oligonucleotide probes of identical sequence to the AT-rich element were reacted with protein extracts from cells cultured under conditions of low and high CO2 atmospheric content. We detected a gel retardation complex of a strong signal intensity in extracts from cells cultured under 15% CO2, but only a weak signal from cells cultured under 1% CO2. Moreover, a DNA affinity precipitation assay identified a 16-kDa protein that bound to nucleotide sequences within the AT-rich element. The partial amino acid sequence of the protein was similar to the deduced protein sequences of ORF129 and ORF155 from Synechocystis 6803. Our findings suggest that the AT-rich element plays a role as a negative CO2-regulatory element and its transacting factor possibly regulates the rbc transcription in response to CO2 levels.
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