Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Mol Syst Biol. 2013 Jun 25;9:678. doi: 10.1038/msb.2013.31.
Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli extracts. While conventional methods for E. coli ribosome reconstitution are non-physiological, our approach attempts to mimic chemical conditions in the cytoplasm, thus permitting several biological processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technology enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties.
纯粹的体外核糖体合成可以为揭示核糖体生物发生的系统生物学、从定义的成分构建最小细胞以及用新功能工程化核糖体提供关键步骤。在这里，作为实现这一目标的第一步，我们报告了一种在粗 S150 大肠杆菌提取物中构建大肠杆菌核糖体的方法。虽然大肠杆菌核糖体重建的传统方法是非生理的，但我们的方法试图模拟细胞质中的化学条件，从而允许同时发生几个生物学过程。具体来说，我们的集成合成、组装和翻译（iSAT）技术能够一步激活 rRNA 转录，将转录的 rRNA 与天然核糖体蛋白组装成功能性核糖体，并使这些核糖体在同一隔室中合成活性蛋白。我们表明，iSAT 通过引入介导对克林霉素抗性的 23S rRNA 突变，使得体外构建修饰的核糖体成为可能。我们预计 iSAT 将有助于核糖体组装的研究，并为具有改变性质的核糖体的制造开辟新途径。
