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Conserved mechanisms for cold adaptation in bacteria and higher plants

细菌和高等植物冷适应的保守机制

基本信息

批准号：
15380231
负责人：
IMAI Ryozo
金额：
$ 8.26万
依托单位：
National Agricultural Research Center for Hokkaido Region, National Agriculture and Bio-oriented Research Ooganization Research Center for Hokkaido Region, National Agriculture and Biooriented Research Organization
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15380231/
关键词：
Cold shock RNA-binding protein RNA chaperone Cold acclimation wheat 分子ビーコン翻訳

项目摘要

Cold shock domain (CSD) protein or Y-box protein is widely distributed in bacteria, plants, and animals. The highly conserved CSD serves as a nucleic acid-binding domain for the functions in translational and/or transcriptional regulations within these proteins. We have identified a cDNA clone from winter wheat (WCSP1) which encodes a protein homologous to E.. coli CspA. The putative WCSP1 protein consists of an N-terminal CSD and three C-terminal CCHC zinc fingers. Both WCSP1 mRNA and protein levels steadily increases in crown tissue during cold acclimation. Recombinant WCSP1 protein is capable of binding RNA and ss/dsDNA. WCSP1 partially complemented a cold sensitive phenotype of E.coli cspA, cspB, cspE, cspG quadruple mutant, suggesting that WCSP1 shares a function with E.coli CSPs for cold adaptation. It has also been demonstrated that WCSP1 showed in vivo RNA chaperone activity in E.coli system. A series of deletion and point mutations were introduced in WCSP1 and the mutant recombinant proteins were purified from E.coli. ssDNA-binding assay revealed that the C-terminal Gly-rich and zinc finger region is not required for ssDNA binding. Point mutations in the RNP I and RNP II RNA recognition motifs within the CSD abolished ssDNA-binding activity. A molecular beacon system with 5'-FITC-labeled and 3'-quencher-labeled oligonucleotides was utilized to demonstrate a double strand nucleic acid melting activity in vitro. The melting activity of WCSP1 and its mutant proteins correlated well with ssDNA binding activity. Therefore it was concluded that RNA chaperone activity of WCSP1 resides within the CSD. Transient expression of WCSP1-GFP protein revealed that WCSP1 is localized in ER and nucleus. The ER localization required the C-terminal zinc finger domain of WCSP1.

冷休克域（Cold shock domain， CSD）蛋白或Y-box蛋白广泛分布于细菌、植物和动物中。高度保守的CSD作为核酸结合域，在这些蛋白的翻译和/或转录调控中发挥作用。我们从冬小麦中鉴定出了一个编码E同源蛋白的cDNA克隆（WCSP1）。杆菌CspA。推定的WCSP1蛋白由一个n端CSD和三个c端CCHC锌指组成。在冷驯化过程中，冠组织中WCSP1 mRNA和蛋白水平均稳定升高。重组WCSP1蛋白能够结合RNA和ss/dsDNA。WCSP1部分补充了大肠杆菌cspA、cspB、cspE、cspG四重突变体的冷敏感表型，表明WCSP1与大肠杆菌CSPs具有冷适应功能。WCSP1在大肠杆菌系统中也表现出体内RNA伴侣活性。在WCSP1中引入了一系列缺失和点突变，并从大肠杆菌中纯化了突变体重组蛋白。ssDNA结合实验显示，c端Gly-rich和锌指区不需要ssDNA结合。CSD内RNP I和RNP II RNA识别基序的点突变破坏了ssdna结合活性。利用5'- fitc标记和3'-quencher标记的寡核苷酸分子信标系统在体外证明双链核酸熔融活性。WCSP1及其突变蛋白的熔解活性与ssDNA结合活性密切相关。由此得出结论，WCSP1的RNA伴侣活性存在于CSD内。瞬时表达WCSP1- gfp蛋白，发现WCSP1定位于内质网和细胞核。内质网定位需要WCSP1的c端锌指结构域。