Szaflarski Witold, Vesper Oliver, Teraoka Yoshika, Plitta Beata, Wilson Daniel N, Nierhaus Knud H
Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Ihnestr. 73, D-14195 Berlin, Germany.
J Mol Biol. 2008 Jun 27;380(1):193-205. doi: 10.1016/j.jmb.2008.04.060. Epub 2008 May 2.
We describe the optimization of a poly(Phe) synthesis system, the conditions of which have been applied for efficient translation of heteropolymeric mRNAs. Here we identify two parameters that are essential to obtain translation at efficiency and accuracy levels equivalent to those in vivo, viz., the fine-tuning of the energy-rich components with an acetyl-phosphate substrate for energy regeneration, as well as the ionic conditions. Applying this system revealed a number of new features: (i) 70S ribosomes are able to recycle within 300 s in a non-enzymatic fashion in the absence of tmRNA. This observation might explain the fact that a knockout of the tmRNA gene ssrA is not lethal for Escherichia coli cells in contrast to other bacterial strains, such as Bacillus subtilis. (ii) The high efficiency of the system was exploited to analyze the misincorporation of various amino acids (resolution limit=1:15,000). No misreading was observed at the middle codon position and only marginal effects were observed at the first one (even when misreading was artificially stimulated 20- to 30-fold), yielding an improved definition of the near-cognate and non-cognate aminoacyl-tRNAs. (iii) Aminoglycosides increase Phe and Lys incorporation about 2-fold in the presence of poly(U) or poly(UUC) and poly(A), respectively, and induce a back-translocation (except hygromycin B) exclusively in the absence of EF-G*GTP, as do the non-related drugs viomycin and edeine.
我们描述了聚（苯丙氨酸）合成系统的优化，该系统的条件已应用于异聚体mRNA的高效翻译。在此，我们确定了两个参数，它们对于在效率和准确性水平上获得与体内相当的翻译至关重要，即利用乙酰磷酸底物对富含能量的成分进行微调以实现能量再生，以及离子条件。应用该系统揭示了许多新特征：（i）在没有tmRNA的情况下，70S核糖体能够以非酶促方式在300秒内循环利用。这一观察结果可能解释了与其他细菌菌株（如枯草芽孢杆菌）不同，tmRNA基因ssrA敲除对大肠杆菌细胞并非致命的事实。（ii）该系统的高效性被用于分析各种氨基酸的错掺入（分辨率极限=1:15,000）。在中间密码子位置未观察到错读，在第一个密码子位置仅观察到边际效应（即使错读被人为刺激20至30倍），从而对近同源和非同源氨酰-tRNA有了更好的定义。（iii）氨基糖苷类药物在分别存在聚（U）或聚（UUC）和聚（A）的情况下，可使苯丙氨酸和赖氨酸的掺入增加约2倍，并且仅在没有EF-G·GTP的情况下诱导反向易位（除潮霉素B外），与非相关药物紫霉素和伊短菌素的作用相同。
