23S rRNA甲基转移酶RrmJ促进细菌50S亚基后期组装

核糖体是所有细胞中负责蛋白质合成的分子机器。它自身在细胞内的组装成熟过程受到严密调控，需要诸多组装因子的参与。RrmJ是原核生物中一类保守的甲基转移酶，能够甲基化修饰核糖体上肽基转移酶中心（peptidyl transferase center, PTC）内A环的U2552位点。敲除rrmJ基因的大肠杆菌表现出显著的生长缺陷及50S亚基组装前体的累积，因而RrmJ在50S亚基组装中具有重要作用。本研究对细菌生长实验与核糖体图谱分析表明，回补表达RrmJ的质粒对于ΔrrmJ菌株生长缺陷有显著改善，50S前体累积现象也得到有效缓解。通过共沉淀实验证明，RrmJ与ΔrrmJ菌株中提取的50S前体结合能力显著强于缺失型或野生型菌株中纯化的成熟50S；当加入S-腺苷甲硫氨酸时，该酶与50S前体结合能力显著下降。冷冻电镜三维重构数据进一步阐明，缺失型菌株50S前体主要停滞在组装晚期两个PTC区域成熟程度不同的特定时段。综合上述结果表明，U2552位点的修饰发生在50S亚基组装晚期特定阶段，这一事件不仅会加速A环的RNA螺旋折叠，另有可能促进附近PTC区域结构成熟。

The Methyltransferase of 23S rRNA RrmJ Accelerates Late-stage Assembly of the 50S Subunit in Escherichia Coli

The ribosome is a universal molecular machine responsible for protein synthesis in all cells. Cellular assembly of ribosomal subunits is tightly regulated and requires several dozens of assembly factors. RrmJ is a methyltransferase highly conserved in prokaryotes, capable of methylating U2552 in A-loop of the peptidyl transferase center (PTC) of the ribosome. The deletion of rrmJ in Escherichia. coli results in growth defects and accumulation of 50S ribosomal subunit precursors, indicating the significance of RrmJ in the 50S assembly. In this study, we found that overexpressing the pBAD-rrmJ plasmids in the ΔrrmJ strain rescued the growth defects and accumulation of 50S precursors. With co-sedimentation assays, we observed that the binding of RrmJ to the 50S precursor particles isolated from ΔrrmJ cells was markedly stronger than the mature 50S purified from the rrmJ deletion mutant or wild type strains. When S-adenosylmethionine was added in the reaction system, the affinity of RrmJ to the 50S precursor dramatically decreased. Cryo-EM structures further indicated that the 50S precursor particles were mainly trapped in two specific late assembly stages with different PTC regions. Together, our data suggest that the modification of U2552 takes place at a specific late stage of the 50S assembly process, and this modification contributes not only to the proper folding of local RNA helices in the A-loop, but also to the conformational maturation of the PTC region.