Abstract：
Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on Escherichia coli growth. Furthermore, we recently constructed a strain with combined KOs of five modification enzymes (RluC, RlmKL, RlmN, RlmM and RluE) of the \'critical region\' of the peptidyl transferase centre (PTC) in 23S rRNA that exhibited only a minor growth defect at 37°C (although major at 20°C). However, our combined KO of modification enzymes RluC and RlmE (not RluE) resulted in conditional lethality (at 20°C). Although the growth rates for both multiple-KO strains were characterized, the molecular explanations for such deficits remain unclear. Here, we pinpoint biochemical defects in these strains. In vitro fast kinetics at 20°C and 37°C with ribosomes purified from both strains revealed, counterintuitively, the slowing of translocation, not peptide bond formation or peptidyl release. Elongation rates of protein synthesis in vivo, as judged by the kinetics of β-galactosidase induction, were also slowed. For the five-KO strain, the biggest deficit at 37°C was in 70S ribosome assembly, as judged by a dominant 50S peak in ribosome sucrose gradient profiles at 5 mM Mg2+. Reconstitution of this 50S subunit from purified five-KO rRNA and ribosomal proteins supported a direct role in ribosome biogenesis of the PTC region modifications per se, rather than of the modification enzymes. These results clarify the importance and roles of the enigmatic rRNA modifications.
摘要：
大核糖体RNA(rRNA)在功能上重要的区域在转录后被大量修饰，但是，矛盾的是,修饰酶的单个敲除（KO）对大肠杆菌生长的影响最小。此外，我们最近构建了一种具有五种修饰酶(RluC，RlmKL,RlmN,23SrRNA中肽基转移酶中心（PTC）的“关键区域”的RlmM和RluE），在37°C时仅表现出轻微的生长缺陷（尽管在20°C时主要）。然而,我们组合的KO修饰酶RluC和RlmE（不是RluE）导致条件致死性（在20°C下）。尽管对两种多KO菌株的生长速率进行了表征，这种缺陷的分子解释尚不清楚.这里,我们确定了这些菌株的生化缺陷。从两种菌株中纯化的核糖体在20°C和37°C下的体外快速动力学显示，反直觉,易位的减慢，不形成肽键或释放肽基。体内蛋白质合成的伸长率，根据β-半乳糖苷酶诱导的动力学判断，也放慢了脚步。对于五KO菌株，37℃时最大的缺陷是70S核糖体组装，如通过在5mMMg2+的核糖体蔗糖梯度谱中的主要50S峰判断。从纯化的5-KOrRNA和核糖体蛋白中重建该50S亚基支持在PTC区域修饰本身的核糖体生物发生中的直接作用，而不是修饰酶。这些结果阐明了神秘的rRNA修饰的重要性和作用。
