大肠杆菌rRNA在36个位置被转录后修饰,但它们的修饰酶对于生长是不必要的,质疑他们的意义。然而,据报道,RlmE酶缺失是一个主要的生长缺陷,消除了23SrRNA的肽基转移酶中心(PTC)附近的2'O甲基化。此外,PTC周围相邻的80-nt“关键区域”必须进行修饰,以在体外产生显着的肽基转移酶活性。令人惊讶的是,我们发现仅缺乏两种rRNA修饰酶是有条件致死的(在20°C):RlmE和RluC。在允许温度(37°C)下,这种双重敲除被证明可以消除四个修饰,并且在核糖体组装中存在缺陷,虽然不超过RlmE单打淘汰赛。然而,双敲除显示出比单一敲除更低的三肽合成率,表明核糖体易位更有缺陷.五种关键区域修饰酶RluC的组合敲除,RlmKL,RlmN,RlmM,和RluE(不是RlmE),合成七个关键区域修饰中的五个以及14个rRNA和tRNA修饰,是可行的(37°C时的轻微生长缺陷,majorat20°C).基于先前的体外研究,这是令人惊讶的。这种五敲除组合在37℃时对核糖体组装和移码的影响很小,但在较冷的温度下对核糖体组装和体外肽基转移酶活性的影响更大。这些结果确立了细菌rRNA修饰酶的条件必要性,并且还揭示了体内PTC区修饰的意外可塑性。
Escherichia coli rRNAs are post-transcriptionally modified at 36 positions but their modification enzymes are dispensable individually for growth, bringing into question their significance. However, a major growth defect was reported for deletion of the RlmE enzyme, which abolished a 2\'O methylation near the peptidyl transferase center (PTC) of the 23S rRNA. Additionally, an adjacent 80-nt \"critical region\" around the PTC had to be modified to yield significant peptidyl transferase activity in vitro. Surprisingly, we discovered that an absence of just two rRNA modification enzymes is conditionally lethal (at 20°C): RlmE and RluC. At a permissive temperature (37°C), this double knockout was shown to abolish four modifications and be defective in ribosome assembly, though not more so than the RlmE single knockout. However, the double knockout exhibited an even lower rate of tripeptide synthesis than did the single knockout, suggesting an even more defective ribosomal translocation. A combination knockout of the five critical-region-modifying enzymes RluC, RlmKL, RlmN, RlmM, and RluE (not RlmE), which synthesize five of the seven critical-region modifications and 14 rRNA and tRNA modifications altogether, was viable (minor growth defect at 37°C, major at 20°C). This was surprising based on prior in vitro studies. This five-knockout combination had minimal effects on ribosome assembly and frameshifting at 37°C, but greater effects on ribosome assembly and in vitro peptidyl transferase activity at cooler temperatures. These results establish the conditional essentiality of bacterial rRNA modification enzymes and also reveal unexpected plasticity of modification of the PTC region in vivo.