Implications of macromolecular crowding and reducing conditions for in vitro ribosome construction.
Implications of macromolecular crowding and reducing conditions for in vitro ribosome construction.
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大分子拥挤和减少体外核糖体构建条件的影响。
DOI：
10.1093/nar/gkv329
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发表时间：
2015-05-19
影响因子：
14.9
通讯作者：
Jewett MC
中科院分区：
文献类型：
--
作者：
Fritz BR;Jamil OK;Jewett MC
大肠杆菌核糖体的体外构建可以阐明对这些复杂分子机器的更深入理解，并使具有新功能的合成变体的生产成为可能。为了实现这一目标，我们最近开发了一个集成的合成，组装和翻译（iSAT）系统，允许共激活的核糖体RNA（rRNA）转录和核糖体组装，mRNA转录和蛋白质翻译没有完整的细胞。在这里，我们发现大分子拥挤和还原剂增加了整体iSAT蛋白合成; 6%w/v Ficoll 400和2 mM DTBA的组合使整体iSAT蛋白合成活性增加了约5倍。通过利用荧光RNA适体、荧光报告蛋白和核糖体沉降分析，我们表明拥挤剂通过增强翻译而增加iSAT产量，而还原剂增加rRNA转录和核糖体组装。最后，我们发现iSAT核糖体具有体内组装的E. coli核糖体。这项工作通过添加拥挤和还原剂改善了iSAT蛋白的合成，提供了对iSAT系统内这些添加剂的影响的透彻理解，并演示了iSAT如何允许在体外转录-翻译系统的背景下操纵和分析核糖体生物合成。
In vitro construction of Escherichia coli ribosomes could elucidate a deeper understanding of these complex molecular machines and make possible the production of synthetic variants with new functions. Toward this goal, we recently developed an integrated synthesis, assembly and translation (iSAT) system that allows for co-activation of ribosomal RNA (rRNA) transcription and ribosome assembly, mRNA transcription and protein translation without intact cells. Here, we discovered that macromolecular crowding and reducing agents increase overall iSAT protein synthesis; the combination of 6% w/v Ficoll 400 and 2 mM DTBA yielded approximately a five-fold increase in overall iSAT protein synthesis activity. By utilizing a fluorescent RNA aptamer, fluorescent reporter proteins and ribosome sedimentation analysis, we showed that crowding agents increase iSAT yields by enhancing translation while reducing agents increase rRNA transcription and ribosome assembly. Finally, we showed that iSAT ribosomes possess ∼70% of the protein synthesis activity of in vivo-assembled E. coli ribosomes. This work improves iSAT protein synthesis through the addition of crowding and reducing agents, provides a thorough understanding of the effect of these additives within the iSAT system and demonstrates how iSAT allows for manipulation and analysis of ribosome biogenesis in the context of an in vitro transcription-translation system.
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影响因子：
3.7
作者：
Ge X;Luo D;Xu J
通讯作者：
Xu J
影响因子：
7.7
作者：
Jewett, Michael C.;Forster, Anthony C.
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Forster, Anthony C.
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HWANG, C;SINSKEY, AJ;LODISH, HF
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LODISH, HF
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Jewett, MC;Swartz, JR
通讯作者：
Swartz, JR
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作者：
Fritz BR;Jewett MC
通讯作者：
Jewett MC