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首页> 外文期刊>Plant physiology >Cis-acting elements and DNA-binding proteins involved in CO2-responsive transcriptional activation of Cah1 encoding a periplasmic carbonic anhydrase Chlamydomonas reinhardtii
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Cis-acting elements and DNA-binding proteins involved in CO2-responsive transcriptional activation of Cah1 encoding a periplasmic carbonic anhydrase Chlamydomonas reinhardtii

机译：Cah1编码周质碳酸酐酶衣藻衣藻的CO2响应转录激活中涉及的顺式作用元件和DNA结合蛋白。

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摘要

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 A (GA) under bar(TT) under barT (C) under bar ACCGGTTGGAAGGAGGT) and EE-2 (C (GA) under barC (TT) under barA (C) under bar GAA). 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 (A (GA) under bar T (TT) under barT (C) under barA) 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.
机译：当细胞在低CO2条件下（0.04％[v / v]）暴露时，会激活莱茵衣藻中编码周质碳酸酐酶的Cah1表达。通过使用芳基硫酸酯酶报告基因，相对于转录起始位点，Cah1转录激活必不可少的调节区被限定为-293和-231之间的63 ​​bp片段。对63 bp区域的接头扫描分析确定了两个增强子元件，在barT（C）下在barT（C）下在bar（TT）下的EE-1 A（GA）和在barC（TT）下在EE-2（C） bar A（C）下的GAA下）。凝胶迁移率迁移分析表明，从在低CO2条件下在光照下生长的细胞中纯化的核提取物含有与EE-1和EE-2特异性相互作用的DNA结合蛋白。使用突变型寡核苷酸探针的凝胶迁移率迁移分析表明，与EE-1结合的蛋白质优先识别EE-1的9 bp序列延伸（在T棒下的T条（TT）在barT上（C在barA下）（A）（GA）称为EEC的保守序列基序GANTTNC，它也存在于EE-2中。 EE-1-和EE-2-结合蛋白在体外与两个增强子中的EEC相互作用。 Cah1的-651至-231的5'上游区域中的四个EEC在低CO2条件下在Cah1的转录激活中起着核心作用。这些EEC结合蛋白甚至存在于高CO2条件（5％[v / v]）下生长的细胞中，或在Cah1未激活时在黑暗中存在。基于这些结果，讨论了Cah1的转录调控与Cah1的5'上游区域中的蛋白质与增强子结合的关系。

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