A Novel Cell-Free Peptide Synthesis Driven by Pyridine

我们已经证明，高浓度（40-60%）吡啶能够促进各种生物体核糖体上的多肽合成，而不需要任何蛋白质因子。为了更好地了解这个吡啶系统的机制，我们利用大肠杆菌核糖体研究了核糖体亚基的结合、核糖体蛋白的组装以及60%吡啶的易位反应。蔗糖密度梯度曲线表明，即使在含20 mM Mg^<2+>的水溶液中，亚基暴露于吡啶也不能重新结合，但在含60%吡啶的水溶液中，它们可以形成总偶对。核糖体蛋白的二维凝胶电泳显示，一些构成大亚基三个特征的蛋白质，特别是那些包含在延伸因子结合结构域（L7/L12 talk和L11）的蛋白质从重组颗粒中释放出来。在60%吡啶中，多苯丙氨酸的合成和易位活性都被新霉素（一种抑制因子依赖性易位的强抑制剂）完全消除，而这两个反应完全不受石膏杆菌（一种抑制因子依赖性易位的核毒素）的影响。一种类似的抗生素抑制剂硫链顿也不影响这两种反应，这与抗生素需要L11才能与核糖体结合的事实是一致的。这些结果表明，易位实际上发生在吡啶系统中，但以一种独立于因素（自发）的方式。

We have already demonstrated that high concentrations (40-60%) of pyridine was able to promote polypeptide synthesis on ribosomes from various organisms without any protein factors. For better understanding the mechanism of this pyridine system, association of ribosomal subunits, assembly of ribosomal proteins, as well as translocation reaction in 60% pyridine were investigated using E.coli ribosomes. Sucrose density gradient profiles showed that while the subunits once exposed to pyridine failed to re-associate themselves in the aqueous solution even containing 20 mM Mg^<2+>, they could form the total couple in 60% pyridine. Two-dimensional gel electrophoresis of ribosomal proteins revealed that some proteins composing the three features of the large subunit, especially those included in the elongation factor-binding domain (the L7/L12 atalk and L11), were released from the reconstituted particle. Both of polyphenylalanine synthesis and translocation activities in 60% pyridine were completely abolished by neomycin, a strong inhibitor against factor-independent translocation, whereas both of the reactions were not affected at all by gypsophilin, a ribotoxin to inhibit factor-dependent translocation. A similar antibiotic inhibitor, thiostrepton, affected neither of the reactions as well, which is consistent with the fact that the antibiotics requires L11 for its binding to the ribosome. These results suggest that translocation actually occurs in the pyridine system, but in a factors-independent (spontaneous) manner.