23S rRNA甲基转移酶RrmJ促进大肠杆菌50S亚基后期组装

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

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。Rrm J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rrm J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Rrm J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Rrm J的质粒对于Δrrm J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,Rrm J与Δrrm J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 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. Rrm J is a methyltransferase highly conserved in prokaryotes,capable of methylating U2552 in Aloop of the peptidyl transferase center（ PTC） of the ribosome. The deletion of rrm J in Escherichia. coli results in growth defects and accumulation of 50 S ribosomal subunit precursors, indicating the significance of Rrm J in the 50 S assembly. In this study,we found that overexpressing the p BAD-rrm J plasmids in the Δrrm J strain rescued the growth defects and accumulation of 50 S precursors. With cosedimentation assays,we observed that the binding of Rrm J to the 50 S precursor particles isolated fromΔrrm J cells was markedly stronger than the mature 50 S purified from the rrm J-deletion mutant or wildtype strains. When S-adenosylmethionine was added in the reaction system,the affinity of Rrm J to the50 S precursor dramatically decreased. Cryo-EM structures further indicated that the 50 S 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 50 S 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.