Fritz Brian R, Jamil Osman K, Jewett Michael C
Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA.
Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA Interdisciplinary Biological Sciences Graduate Program, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA Northwestern Institute on Complex Systems, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA Simpson Querrey Institute, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA Chemistry of Life Processes Institute, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA
Nucleic Acids Res. 2015 May 19;43(9):4774-84. doi: 10.1093/nar/gkv329. Epub 2015 Apr 20.
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.
体外构建大肠杆菌核糖体能够更深入地理解这些复杂的分子机器，并使生产具有新功能的合成变体成为可能。为了实现这一目标，我们最近开发了一种整合合成、组装和翻译（iSAT）系统，该系统能够在没有完整细胞的情况下共同激活核糖体RNA（rRNA）转录和核糖体组装、mRNA转录和蛋白质翻译。在这里，我们发现大分子拥挤效应和还原剂可提高iSAT的整体蛋白质合成；6% w/v的聚蔗糖400和2 mM二硫苏糖醇双乙酸酯（DTBA）的组合使iSAT的整体蛋白质合成活性提高了约五倍。通过使用荧光RNA适配体、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来提高iSAT产量，而还原剂则增加rRNA转录和核糖体组装。最后，我们表明iSAT核糖体具有体内组装的大肠杆菌核糖体约70%的蛋白质合成活性。这项工作通过添加拥挤剂和还原剂提高了iSAT蛋白质合成，全面了解了这些添加剂在iSAT系统中的作用，并展示了iSAT如何在体外转录-翻译系统的背景下对核糖体生物发生进行操作和分析。
