Molecular mechanism of protein synthesis

我们发现，高浓度（40-60%）的吡啶（一种芳香叔胺催化剂）能够促进核糖体上的翻译，而不需要化学能量来源的可溶性蛋白质因子。这种新的翻译体系被称为吡啶体系，根据相应的模板，poly (U)和poly (A)，它可以产生链长高达十聚体的寡苯丙氨酸和链长约40聚体的聚赖氨酸。在聚（UC）依赖性低聚物（丝氨酸-亮氨酸）合成中，丝氨酸和亮氨酸交替序列的寡肽是主要产物。模板依赖性在吡啶含量为60%时最为突出，但在吡啶含量为40%或50%时则没有观察到。该反应的最佳温度范围为30 ~ 60℃，这可能反映了该反应不需要对温度敏感的可溶性蛋白因子。吡啶体系中K^+ (200 ~ 500 mM)和Mg^<2+> (1 ~ 50 mM)的最佳浓度范围也较宽，与一般的水平移体系有很大的不同。目前的研究表明，rRNAs在核糖体功能中起着核心作用，尽管尚未提供确凿的证据；一些论文报道了真细菌的肽基转移，即在核糖体大亚基上形成肽键，几乎不需要它们的蛋白质。本研究证明，无论是从核糖体中提取的还是体外合成的大肠杆菌23S rrna，在40%吡啶存在的情况下，都能够在没有模板的情况下促进肽键的形成（由苯丙酰- trna ^<Phe>合成苯丙酰-苯丙氨酸）。该反应需要23S rRNA或处于折叠状态的转录本，氯霉素抑制了该反应，但环己亚胺抑制不了该反应，23S rRNA的RNase H与一些互补的寡脱氧核糖核苷酸杂交也抑制不了该反应。这些结果有力地表明23S rRNA直接参与肽键的形成。少

We found that a high concentration (40-60%) of pyridine, an aromatic tertiary amine catalyst, is able to promote the translation on ribosomes without soluble protein factors of chemical energy sources. This novel translation system was called the pyridine system, which could produce oligophyenylalanine with chain lengths of up to decamer and polylysine with chain lengths of around 40mer, depending on the corresponding templates, poly (U) and poly (A), respectively. In poly (UC) -dependent oligo (serine-leucine) synthesis, oligopeptides with a serine and leucine alternate sequence were the main products. The template dependency is most prominent at 60% pyridine, but not observed at 40% or 50% pyridine. The optimal temperature range of the reaction was rather wide between 30 and 60ﾟC,which may reflect no requirement for temperature-sensitive soluble protein factors for the reaction. The ranges of the optimal concentrations of K^+ (200-500 mM) and Mg^<2+> (1-50 mM) in the pyridine system … More are also rather wide, quite different from those in the usual aqueous translation system.Current investigations have suggested that rRNAs play a central role in the ribosomal functions, although the conclusive evidence has not been provided ; some papers have reported that the eubacterial peptidyltransferation, the peptide-bond formation on the ribosomal large subunits, scarcely requires their proteins. Here we demonstrate that E.coli 23S rRNAs either extracted from ribosomes or synthesized in vitro are able to promote the peptide-bond formation (phenylalanylphenylalanine synthesis from phenylalanyl-tRNA^<Phe>) without template in the presence of 40% pyridine. The reaction required 23S rRNAs or the transcripts in the folded state, and was inhibited with chloramphenicol but not with cycloheximide as well as by site-directed digestion with RNase H of 23S rRNA hybridized with some complementary oligodeoxyribonucleotides. These results strongly suggest that 23S rRNA is directly involved in the peptide bond formation. Less