The effect of macromolecular crowding on single-round transcription by Escherichia coli RNA polymerase
The effect of macromolecular crowding on single-round transcription by Escherichia coli RNA polymerase
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DOI：
10.1093/nar/gky1277
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发表时间：
2019-02-20
影响因子：
14.9
通讯作者：
Weiss, Shimon
中科院分区：
文献类型：
--
作者：
Chung, SangYoon;Lerner, Eitan;Weiss, Shimon
以前的工作已经报道了大分子拥挤对生物分子的结构和行为的显着影响。与体外缓冲溶液相反，拥挤的细胞内环境可能对生物分子产生类似的影响。RNA聚合酶（RNAP）作为基因组的看门人，是受调控最多的酶之一。虽然以前证明了大分子拥挤会影响RNAP与DNA的结合，但对拥挤如何作用于晚期起始和启动子清除步骤知之甚少，这些步骤被认为是许多启动子的速率决定步骤。在这里，我们证明了大分子拥挤提高了速率的后期启动和启动子清除使用体外猝灭为基础的单分子动力学测定。此外，增强的拥挤的大小的依赖显着偏离预测的缩放粒子理论，通常用于描述拥挤效应。我们的发现揭示了酶反应如何受到细胞环境中拥挤条件的影响。
Previous works have reported significant effects of macromolecular crowding on the structure and behavior of biomolecules. The crowded intracellular environment, in contrast to in vitro buffer solutions, likely imparts similar effects on biomolecules. The enzyme serving as the gatekeeper for the genome, RNA polymerase (RNAP), is among the most regulated enzymes. Although it was previously demonstrated that macromolecular crowding affects association of RNAP to DNA, not much is known about how crowding acts on late initiation and promoter clearance steps, which are considered to be the rate-determining steps for many promoters. Here, we demonstrate that macromolecular crowding enhances the rate of late initiation and promoter clearance using in vitro quenching-based single-molecule kinetics assays. Moreover, the enhancement's dependence on crowder size notably deviates from predictions by the scaled-particle theory, commonly used for description of crowding effects. Our findings shed new light on how enzymatic reactions could be affected by crowded conditions in the cellular milieu.