Data_Sheet_1_Single-molecule dynamics suggest that ribosomes assemble at sites of translation in Bacillus subtilis.pdf

Eukaryotic cells transcribe ribosomal RNA and largely assemble ribosomes in a structure called the nucleolus, where chromosomal regions containing rRNA operons are clustered. In bacteria, many rRNA operons cluster close to the origin regions that are positioned on the outer borders of nucleoids, close to polar areas, where translating 70S ribosomes are located. Because outer regions of the nucleoids contain the highest accumulation of RNA polymerase, it has been hypothesized that bacteria contain “nucleolus-like” structures. However, ribosome subunits freely diffuse through the entire cells, and could thus be assembled and matured throughout the non-compartmentalized cell. By tracking single molecules of two GTPases that play an essential role in ribosomal folding and processing in Bacillus subtilis, we show that this process takes place at sites of translation, i.e., predominantly at the cell poles. Induction of the stringent response led to a change in the population of GTPases assumed to be active in maturation, but did not abolish nucleoid occlusion of ribosomes or of GTPases. Our findings strongly support the idea of the conceptualization of nucleolus-like structures in bacteria, i.e., rRNA synthesis, ribosomal protein synthesis and subunit assembly occurring in close proximity at the cell poles, facilitating the efficiency of ribosome maturation even under conditions of transient nutrient deprivation.

真核细胞会转录核糖体RNA（ribosomal RNA, rRNA），并主要在名为核仁（nucleolus）的结构中完成核糖体的组装，携带核糖体RNA操纵子（rRNA operons）的染色体区域即聚集于此。在细菌中，多数核糖体RNA操纵子聚集于定位于类核（nucleoid）外缘的复制起始区域（origin regions）附近，该区域紧邻细胞极区，亦是正在进行翻译的70S核糖体（70S ribosome）的所在位置。由于类核外缘区域聚集了最多的RNA聚合酶（RNA polymerase），因此有假说提出细菌中存在‘类核仁结构’。然而，核糖体亚基可在整个细胞内自由扩散，因此核糖体的组装与成熟过程本可在无膜区室化的细菌细胞内全域发生。本研究通过追踪枯草芽孢杆菌（Bacillus subtilis）中两种对核糖体折叠与加工至关重要的GTP酶（GTPases）的单分子运动，证实核糖体成熟与组装过程发生于翻译位点，即主要集中于细胞极区。严谨反应（stringent response）的诱导会改变被认为参与核糖体成熟过程的GTP酶群体分布，但并未消除核糖体与GTP酶的类核阻隔（nucleoid occlusion）现象。本研究结果有力支持了细菌中存在类核仁结构的概念：即核糖体RNA合成、核糖体蛋白合成以及亚基组装均在细胞极区附近紧密发生，即便在短暂营养匮乏的条件下，也能提升核糖体成熟的效率。