Cis-acting elements and DNA-binding proteins involved in CO2-responsive transcriptional activation of Cah1 encoding a periplasmic carbonic anhydrase in Chlamydomonas reinhardtii.
Expression of Cah1, encoding a periplasmic carbonic anhydrase in Chlamydomonas reinhardtii Dangeard, is activated when cells are exposed to low-CO2 conditions (0.04% [v/v]) in light. By using an arylsulfatase reporter gene, a regulatory region essential for the transcriptional activation of Cah1 was delimited to a 63-bp fragment between -293 and -231 relative to the transcription start site. Linker-scan analysis of the 63-bp region identified two enhancer elements, EE-1 (AGATTTTCACCGGTTGGAAGGAGGT) and EE-2 (CGACTTACGAA). Gel mobility shift assays indicated that nuclear extracts purified from cells grown under low-CO2 conditions in light contained DNA-binding proteins specifically interacting with EE-1 and EE-2. Gel mobility shift assays using mutant oligonucleotide probes revealed that the protein binding to EE-1 preferentially recognized a 9-bp sequence stretch (AGATTTTCA) of EE-1, containing a conserved sequence motif named EEC, GANTTNC, which is also present in EE-2. The EE-1- and EE-2-binding proteins interacted with the EECs contained in both of the two enhancer elements in vitro. Four EECs in the 5'-upstream region from -651 to -231 of Cah1 played a central role in the transcriptional activation of Cah1 under low-CO2 conditions. These EEC-binding proteins were present even in cells grown under high-CO2 conditions (5% [v/v]) or in the dark when Cah1 is not activated. On the basis of these results, the relationship between the transcriptional regulation of Cah1 and protein-binding to the enhancer elements in the 5'-upstream region of Cah1 is discussed.
Kucho
Ken-ichi
K
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
Yoshioka
Satoshi
S
Taniguchi
Fumiya
F
Ohyama
Kanji
K
Fukuzawa
Hideya
H
D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D002245:Carbon Dioxide / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000235:genetics*; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003624:Darkness; D004742:Enhancer Elements, Genetic; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics* / Q000528:radiation effects; D008027:Light; D008969:Molecular Sequence Data; D015533:Transcriptional Activation
D017931:DNA Primers; D004268:DNA-Binding Proteins; D002245:Carbon Dioxide; D004247:DNA; D002256:Carbonic Anhydrases