Kinetic cooperativity in Escherichia coli 30S ribosomal subunit reconstitution reveals additional complexity in the assembly landscape
Kinetic cooperativity in Escherichia coli 30S ribosomal subunit reconstitution reveals additional complexity in the assembly landscape
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DOI：
10.1073/pnas.0912007107
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发表时间：
2010-03-23
影响因子：
11.1
通讯作者：
Williamson, James R.
中科院分区：
文献类型：
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作者：
Bunner, Anne E.;Beck, Andrea H.;Williamson, James R.
大肠杆菌 30S 核糖体亚基在体外以分层方式自组装，在平衡条件下，蛋白质与 RNA 的结合是通过其他蛋白质的先前结合实现的。早期 16S rRNA 结合蛋白也比晚期结合蛋白结合得更快，但晚期蛋白结合缓慢的具体原因仍不清楚。此前，开发了一种通过定量质谱法监测的脉冲追踪，用于监测 30S 亚基组装动力学，这里使用修改后的实验方案来探测动力学协同性，包括在脉冲追踪动力学之前包括核糖体蛋白子集结合并启动组装的步骤。在这项工作中，30S 核糖体亚基动力学重建实验表明，热力学依赖性并不总是与动力学协同性相关。一些导致随后的蛋白质结合更加积极有利的折叠转变不会导致更快的蛋白质结合。尽管 3' 结构域初级蛋白 S7 是蛋白质 S9 和 S19 两者的 RNA 结合所必需的，但 S7 的先前结合会加速 S9 的结合，但不会加速 S19 的结合，表明 S19 的结合需要额外的机制步骤。这些关于动力学协同性和多相组装动力学存在的数据揭示了以前隐藏的组装景观的复杂性。
The Escherichia coli 30S ribosomal subunit self-assembles in vitro in a hierarchical manner, with the RNA binding by proteins enabled by the prior binding of others under equilibrium conditions. Early 16S rRNA binding proteins also bind faster than late-binding proteins, but the specific causes for the slow binding of late proteins remain unclear. Previously, a pulse-chase monitored by quantitative mass spectrometry method was developed for monitoring 30S subunit assembly kinetics, and here a modified experimental scheme was used to probe kinetic cooperativity by including a step where subsets of ribosomal proteins bind and initiate assembly prior to the pulse-chase kinetics. In this work, 30S ribosomal subunit kinetic reconstitution experiments revealed that thermodynamic dependency does not always correlate with kinetic cooperativity. Some folding transitions that cause subsequent protein binding to be more energetically favorable do not result in faster protein binding. Although 3' domain primary protein S7 is required for RNA binding by both proteins S9 and S19, prior binding of S7 accelerates the binding of S9, but not S19, indicating there is an additional mechanistic step required for S19 to bind. Such data on kinetic cooperativity and the presence of multiphasic assembly kinetics reveal complexity in the assembly landscape that was previously hidden.