Murphy L D, Zimmerman S B
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Biochim Biophys Acta. 1994 Oct 18;1219(2):277-84. doi: 10.1016/0167-4781(94)90049-3.
DNA-binding protein fractions from exponential and stationary phase cell extracts of E. coli were isolated by affinity chromatography on native DNA-cellulose. The ability of these fractions to convert DNA into a readily-sedimented form was compared in the absence or presence of added polymers. In the absence of polymers, large amounts of the proteins were required. In the presence of polyethylene glycol or polyvinylpyrrolidone, much smaller amounts of the DNA-binding proteins were required, indicating a macromolecular crowding effect from these polymers. The enhanced binding under crowded conditions appears to resolve a paradox between the cellular abundance of the DNA-binding proteins and the amounts required in earlier in vitro studies. The 'histone-like' protein HU from the DNA-binding protein fraction was preferentially incorporated into the pelleted DNA in the presence of polymers. Purified HU at roughly similar amounts caused a similar conversion of DNA to a readily-sedimentable ('condensed') form. Crowding-enhancement of DNA condensation by promoting the binding of proteins to the DNA provides a model for the stabilization of systems such as the bacterial nucleoid or kinetoplast DNA.
通过在天然DNA-纤维素上进行亲和层析，从大肠杆菌指数生长期和稳定期细胞提取物中分离出DNA结合蛋白组分。在添加或不添加聚合物的情况下，比较了这些组分将DNA转化为易于沉降形式的能力。在没有聚合物的情况下，需要大量的蛋白质。在聚乙二醇或聚乙烯吡咯烷酮存在的情况下，所需的DNA结合蛋白量要少得多，这表明这些聚合物具有大分子拥挤效应。在拥挤条件下增强的结合似乎解决了DNA结合蛋白在细胞中的丰度与早期体外研究中所需量之间的矛盾。在聚合物存在的情况下，DNA结合蛋白组分中的“类组蛋白”蛋白HU优先掺入沉淀的DNA中。大致相同量的纯化HU会使DNA发生类似的转化，形成易于沉降的（“凝聚”）形式。通过促进蛋白质与DNA的结合来增强拥挤对DNA凝聚的作用，为细菌类核或动质体DNA等系统的稳定提供了一个模型。
