Functional conservation of cold shock domains in bacteria and higher plants
Functional conservation of cold shock domains in bacteria and higher plants
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DOI：
10.1073/pnas.0603168103
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发表时间：
2006-06-27
影响因子：
11.1
通讯作者：
Imai, Ryozo
中科院分区：
文献类型：
--
作者：
Nakaminami, Kentaro;Karlson, Dale T.;Imai, Ryozo
在大肠杆菌中，冷休克蛋白（CSP）家族在低温下通过破坏RNA二级结构而发挥转录抗终止子或翻译增强子的功能。小麦核酸结合蛋白（WCSP 1）含有一个冷激结构域（CSD），与E.大肠杆菌冷休克蛋白。在本研究中，一系列的突变被引入到WCSP 1，并通过使用在体内和体外试验的背景下，与E. coli CSP。WT WCSP 1在E. colicspA、cspB、cspE、cspG四重缺失突变体补充了其冷敏感表型，表明WCSP 1与大肠杆菌具有相同的功能。coli CSP进行冷适应。此外，通过使用E.在氯霉素抗性基因上游具有发夹环的大肠杆菌菌株。体外dsDNA熔解试验清楚地表明，WCSP 1熔解dsDNA，该活性与结合ssDNA的能力正相关。当在WCSP 1的共有RNA结合基序（RNP 1和RNP 2）内的关键残基处引入突变时，它不能解链dsDNA。WCSP 1-GFP融合蛋白的研究记录了与ER和核定位一致的模式。在体内和体外功能分析，加上亚细胞定位数据，表明WCSP 1可能作为一个RNA分子伴侣，不稳定的二级结构，并参与在低温下的翻译调节。
In Escherichia coli, a family of cold shock proteins (CSPs) function as transcription antiterminators or translational enhancers at low temperature by destabilizing RNA secondary structure. A wheat nucleic acid-binding protein (WCSP1) was found to contain a cold shock domain (CSD) bearing high similarity to E. coli cold shock proteins. In the present study, a series of mutations were introduced into WCSP1, and its functionality was investigated by using in vivo and in vitro assays in the context of functional conservation with E. coli CSPs. Constitutive expression of WT WCSP1 in an E. coli cspA, cspB, cspE, cspG quadruple deletion mutant complemented its cold-sensitive phenotype, suggesting that WCSP1 shares a function with E. coli CSPs for cold adaptation. In addition, transcription antitermination activity was demonstrated for WCSP1 by using an E. coli strain that has a hairpin loop upstream of a chloramphenicol resistance gene. In vitro dsDNA melting assays clearly demonstrated that WCSP1 melts dsDNA, an activity that was positively correlated to the ability to bind ssDNA. When mutations were introduced at critical residues within the consensus RNA binding motifs (RNP1 and RNP2) of WCSP1, it failed to melt dsDNA. Studies with WCSP1-GFP fusion proteins documented patterns that are consistent with ER and nuclear localization. In vivo and in vitro functional analyses, coupled with subcellular localization data, suggest that WCSP1 may function as a RNA chaperone to destabilize secondary structure and is involved in the regulation of translation under low temperature.