Mutational characterization and mapping of the 70S ribosome active site
Mutational characterization and mapping of the 70S ribosome active site
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DOI：
10.1093/nar/gkaa001
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发表时间：
2020-03-18
影响因子：
14.9
通讯作者：
Jewett, Michael C.
中科院分区：
文献类型：
--
作者：
d'Aquino, Anne E.;Azim, Tasfia;Jewett, Michael C.
大肠杆菌核糖体的合成能力吸引了人们对其重新利用以实现新功能的努力，例如合成含有非天然结构单元的聚合物。然而，由于缺乏完整的肽基转移酶中心（PTC）活性位点突变分析来指导设计，重新利用核糖体的努力受到限制。为了解决这一限制，我们利用体外核糖体合成平台来构建和测试 PTC 环、A 环和 P 环内每一个可能的单核苷酸突变，总共 180 个点突变。通过评估大量蛋白质合成动力学、通读、组装和结构图谱来表征这些突变核糖体。尽管 PTC 具有高度保守的性质，但我们发现 > 85% 的 PTC 核苷酸具有突变灵活性。我们的工作代表了 70S 核糖体活性位点的全面单点突变表征和绘图。我们预计它将促进核糖体内的结构-功能关系，并使新的合成生物学应用成为可能。
The synthetic capability of the Escherichia coli ribosome has attracted efforts to repurpose it for novel functions, such as the synthesis of polymers containing non-natural building blocks. However, efforts to repurpose ribosomes are limited by the lack of complete peptidyl transferase center (PTC) active site mutational analyses to inform design. To address this limitation, we leverage an in vitro ribosome synthesis platform to build and test every possible single nucleotide mutation within the PTC-ring, A-loop and P-loop, 180 total point mutations. These mutant ribosomes were characterized by assessing bulk protein synthesis kinetics, readthrough, assembly, and structure mapping. Despite the highly-conserved nature of the PTC, we found that >85% of the PTC nucleotides possess mutational flexibility. Our work represents a comprehensive single-point mutant characterization and mapping of the 70S ribosome's active site. We anticipate that it will facilitate structure-function relationships within the ribosome and make possible new synthetic biology applications.