Identification and purification of a spinach chloroplast DNA-binding protein that interacts specifically with the plastid psaA-psaB-rps14 promoter region
Identification and purification of a spinach chloroplast DNA-binding protein that interacts specifically with the plastid psaA-psaB-rps14 promoter region
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DOI：
10.1007/s004250050203
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发表时间：
1997-11-01
期刊：
影响因子：
4.3
通讯作者：
Chen, SCG
中科院分区：
文献类型：
--
作者：
Cheng, MC;Wu, SP;Chen, SCG
我们以前已经表明，在叶绿体中存在的序列特异性DNA结合蛋白，与位于下游和上游的质体psaA-psaB-rps 14操纵子的5 '-转录起始位点的两个区域特异性相互作用。为了阐明植物发育过程中质体转录的调控机制，本文报道了菠菜叶绿体DNA结合蛋白的纯化和鉴定。变种spinosa Ashers et Graeden）叶，其特异性识别相对于转录起始位点的+64至+83位置之间的序列。这种DNA结合蛋白已高度纯化叶绿体通过使用高盐提取，硫酸铵沉淀，肝素-琼脂糖层析，和序列特异性DNA亲和层析的组合。基于凝胶过滤，该蛋白质表现出59-60 kDa的表观分子量。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，然后西南印迹分析进一步表明，这种DNA结合蛋白是二聚体，由两个近似31 kDa的亚基。我们讨论了这种蛋白质的性质与已知的叶绿体DNA结合因子的质体基因表达。
We have previously shown the presence in chloroplasts of sequence-specific DNA-binding proteins that interact specifically with two regions located downstream and upstream from the 5'-transcription start site of the plastid psaA-psaB-rps14 operon. As part of an effort to elucidate the regulatory mechanism of plastid transcription during plant development, we report here the purification and characterization of the chloroplast DNA-binding protein from spinach (Spinacia oleracea L. var. spinosa Ashers et Graeden) leaves that specifically recognizes sequences between positions +64 to +83 relative to the transcription start site. This DNA-binding protein has been highly purified from chloroplasts by using a combination of high-salt extraction, ammonium sulfate precipitation, heparin-agarose chromatography, and sequence-specific DNA-affinity chromatography. The protein exhibited an apparent molecular weight of 59-60 kDa on the basis of gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Southwestern blot analysis further indicated that this DNA-binding protein is dimeric and composed of two approximate to 31-kDa subunits. We discuss the properties of this protein in relation to the known chloroplast DNA-binding factors for plastid gene expression.