Journal Article

CO<sub>2</sub> Response Element and Corresponding <i>trans-</i>acting Factor of the Promoter for Ribulose-1,5-bisphosphate Carboxylase/oxygenase Genes in <i>Synechococcus</i> sp. PCC7002 Found by an Improved Electrophoretic Mobility Shift Assay

Takuo Onizuka, Hideo Akiyama, Sumiyo Endo, Shozo Kanai, Masahiko Hirano, Satoshi Tanaka and Hitoshi Miyasaka

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 43, issue 6, pages 660-667
Published in print June 2002 | ISSN: 0032-0781
Published online June 2002 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcf082
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

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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 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 trans-acting factor possibly regulates the rbc transcription in response to CO2 levels.

Keywords: Keywords: CO2-regulatory element — DNA-binding proteins — Ribulose-1,5-bisphosphate carboxylase/oxygenase — Synechococcus sp. PCC7002 (Agmenellum quadruplicatum PR-6).; Abbreviations: 6-FAM, 6-carboxyfluorescein; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; ORF, open reading frame; RT-PCR, reverse transcriptional polymerase chain reaction; rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase.

Journal Article.  4763 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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