Conserved mechanisms for cold adaptation in bacteria and higher plants

冷休克结构域(CSD)蛋白或Y-box蛋白广泛存在于细菌、植物和动物体内。高度保守的CSD作为核酸结合域，参与这些蛋白质的翻译和/或转录调节功能。我们已经从冬小麦中克隆到一个编码与E.ColiCSPA。推测的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，WCSP1基因和蛋白在树冠组织中的表达水平稳步上升。重组WCSP1蛋白能与RNA和ss/dsDNA结合。WCSP1部分互补了大肠杆菌CSPA、CSPB、CSPE、CSPG四重突变体的冷敏感表型，表明WCSP1与E.ColiCSPs具有共同的冷适应功能。研究还表明，WCSP1在大肠杆菌系统中具有活体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型重组蛋白。单链DNA结合分析表明，C端富含甘氨酸和锌指区域不是单链DNA结合所必需的。CSD内RNP I和RNP II RNA识别基序的点突变取消了ssDNA结合活性。用5‘-FITC标记和3’-猝灭剂标记的寡核苷酸组成的分子信标系统，在体外显示了双链核酸的熔融活性。WCSP1及其突变蛋白的熔融活性与单链DNA结合活性有很好的相关性。因此，WCSP1的RNA伴侣活性位于CSD内。WCSP1-GFP蛋白的瞬时表达表明WCSP1定位于内质网和胞核。内质网定位需要WCSP1的C-末端锌指结构域。

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.