Department of Cell and Molecular Biology, Uppsala University, Uppsala 75124, Sweden.
Department of Molecular Biology, University of Tartu, 51010 Tartu, Estonia.
RNA. 2022 Jun;28(6):796-807. doi: 10.1261/rna.079096.121. Epub 2022 Mar 8.
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' 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.
rRNAs 在 36 个位置上发生转录后修饰，但它们的修饰酶单独缺失并不影响生长，这让人质疑它们的重要性。然而，RlmE 酶的缺失会导致一个主要的生长缺陷，该酶会使靠近肽基转移酶中心（PTC）的 2' 位甲基化。此外，PTC 周围的一个相邻的 80nt“关键区域”必须进行修饰，才能在体外产生显著的肽基转移酶活性。令人惊讶的是，我们发现仅缺少两种 rRNA 修饰酶是有条件致死的（在 20°C 时）：RlmE 和 RluC。在允许的温度（37°C）下，这种双敲除被证明会消除四个修饰，并使核糖体组装缺陷，但不如 RlmE 单敲除严重。然而，双敲除的三肽合成率甚至比单敲除更低，这表明核糖体易位的缺陷更大。五个关键区域修饰酶 RluC、RlmKL、RlmN、RlmM 和 RluE（不包括 RlmE）的组合敲除，它们总共合成五个关键区域修饰中的五个和 14 个 rRNA 和 tRNA 修饰，是可行的（在 37°C 时有轻微的生长缺陷，在 20°C 时则有较大的缺陷）。这与之前的体外研究结果出人意料。这种五敲除组合对 37°C 时的核糖体组装和移码影响较小，但对核糖体组装和体外肽基转移酶活性的影响较大。这些结果确立了细菌 rRNA 修饰酶的条件必需性，也揭示了 PTC 区域修饰在体内的出乎意料的可塑性。
