INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS

大分子拥挤对生化系统的影响

• 批准号: 5201983
• 负责人: S B ZIMMERMAN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5201983
• 关键词: DNA binding protein; Escherichia coli; bacterial DNA; biophysics; cell nucleus; chemical aggregate; chemical condensation; chemical stability; chromosomes; conformation; cytoplasm; intermolecular interaction; solutions; thermodynamics

The DNA of the bacterial chromosome is localized ina compact body called the nucleoid. Bacterial nucleoids have been extensively studied, but the basis of nucleoid stabilization has remained elusive. The nucleoid is immersed in a cytoplasm which is very concentrated in macromolecules, giving a potential for large macromolecular crowding effects. The following results suggest that crowding-enhancement of the interaction between DNA-binding proteins and the cellular DNA may provide an important stabilizing force for the compact form of DNA in the bacterial nucleoid. These results include the first use of concentrated cell extracts to supply a crowded environment in model studies of the mechanisms of DNA compaction within bacterial or other cells. DNA added to concentrated extracts of Escherichia coli undergoes a reversible transition to a readily-sedimentable ("condensed") form. The extract plays two roles in this transition, supplying both DNA-binding protein(s) and a crowded environment that increases protein binding and favors compact DNA conformations. The two roles are based on properties of fractions prepared by absorption of extracts with DNA-cellulose, and are consistent with model studies of condensation by combinations of purified DNA-binding materials (protein HU or spermidine) and concentrated solutions of crowding agents (albumin or PEG 8000); in each case, crowding agents and DNA-binding materials jointly reduced the amounts of each other required for condensation. Condensation of the added DNA molecules caused large increases in the rate of cohesion between their complementary single-stranded termini. Cohesion products of lambda DNA made in vitro are a mixture of linear and circular aggregates, whereas those made in vivo are cyclic monomers. We suggest a simple mechanism based upon consensation at the site of viral injection which may explain this discrepancy.

细菌染色体的DNA位于一个紧凑的身体， 类核 细菌类核已被广泛研究，但 类核稳定化的基础仍然难以捉摸。 类核是 沉浸在细胞质中，细胞质中的大分子非常集中， 从而可能产生大的大分子拥挤效应。 的 以下结果表明，拥挤增强的相互作用 DNA结合蛋白和细胞DNA之间的相互作用可以提供 细菌中DNA紧密形式的重要稳定力 类核 这些结果包括第一次使用浓缩细胞 提取物，以提供一个拥挤的环境中的模型研究的 细菌或其他细胞内的DNA压缩机制。 加入到大肠杆菌浓缩提取物中的DNA经历了 可逆地转变为易于沉淀（“凝聚”）的形式。 的 提取物在这种转变中起着两个作用，既提供DNA结合， 蛋白质和拥挤的环境，增加蛋白质结合， 有利于紧凑的DNA构象。 这两个角色基于属性 通过用DNA-纤维素吸收提取物制备的级分，和 与模型研究的冷凝相一致， 纯化的DNA结合材料（蛋白质HU或亚精胺）和 拥挤剂（白蛋白或PEG 8000）的浓缩溶液;在每一个 在这种情况下，拥挤剂和DNA结合材料共同降低了 彼此之间的量是冷凝所需的。 加入的DNA分子的凝聚导致了DNA分子的大量增加。 其互补单链末端之间的结合率。 体外制备的λ DNA的内聚产物是线性和非线性的混合物。 环状聚集体，而体内产生的那些是环状单体。 我们 提出了一个简单的机制，基于在病毒感染部位的共识， 这可能解释了这种差异。