CO(2) 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.
We analyzed the promoter of the genes encoding the ribulose-1,5-bisphosphate carboxylase/oxygenase (rbc) in the cyanobacterium Synechococcus sp. PCC7002 and localized the CO(2)-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% CO(2) conditions. We found that the AT-rich element localized from -262 to -291 relative to the rbc translation-starting site was required for CO(2)-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 CO(2) atmospheric content. We detected a gel retardation complex of a strong signal intensity in extracts from cells cultured under 15% CO(2), but only a weak signal from cells cultured under 1% CO(2). 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 CO(2)-regulatory element and its trans-acting factor possibly regulates the rbc transcription in response to CO(2) levels.
Onizuka
Takuo
T
Biological Sciences Department, Toray Research Center, Inc., 1111 Tebiro, Kamakura, Kanagawa, 248-8555 Japan. Takuo_Onizuka@trc.toray.co.jp
Akiyama
Hideo
H
Endo
Sumiyo
S
Kanai
Shozo
S
Hirano
Masahiko
M
Tanaka
Satoshi
S
Miyasaka
Hitoshi
H
D000251:Adenosine Triphosphatases / Q000187:drug effects / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002245:Carbon Dioxide / Q000494:pharmacology*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D000458:Cyanobacteria / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020218:Response Elements / Q000235:genetics*; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics* / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D015534:Trans-Activators; D002245:Carbon Dioxide; D015500:Chloramphenicol O-Acetyltransferase; D000251:Adenosine Triphosphatases; C101625:RbcL protein, plastid; D012273:Ribulose-Bisphosphate Carboxylase