Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA.
Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
Nucleic Acids Res. 2023 Feb 28;51(4):1927-1942. doi: 10.1093/nar/gkad047.
Ribosomes of Bacteroidia (formerly Bacteroidetes) fail to recognize Shine-Dalgarno (SD) sequences even though they harbor the anti-SD (ASD) of 16S rRNA. Inhibition of SD-ASD pairing is due to sequestration of the 3' tail of 16S rRNA in a pocket formed by bS21, bS18, and bS6 on the 30S platform. Interestingly, in many Flavobacteriales, the gene encoding bS21, rpsU, contains an extended SD sequence. In this work, we present genetic and biochemical evidence that bS21 synthesis in Flavobacterium johnsoniae is autoregulated via a subpopulation of ribosomes that specifically lack bS21. Mutation or depletion of bS21 in the cell increases translation of reporters with strong SD sequences, such as rpsU'-gfp, but has no effect on other reporters. Purified ribosomes lacking bS21 (or its C-terminal region) exhibit higher rates of initiation on rpsU mRNA and lower rates of initiation on other (SD-less) mRNAs than control ribosomes. The mechanism of autoregulation depends on extensive pairing between mRNA and 16S rRNA, and exceptionally strong SD sequences, with predicted pairing free energies of < -13 kcal/mol, are characteristic of rpsU across the Bacteroidota. This work uncovers a clear example of specialized ribosomes in bacteria.
拟杆菌门（原拟杆菌门）的核糖体无法识别 Shine-Dalgarno (SD) 序列，尽管它们携带有 16S rRNA 的反 SD (ASD)。SD-ASD 配对的抑制是由于 16S rRNA 的 3' 尾巴被 30S 平台上的 bS21、bS18 和 bS6 形成的口袋隔离。有趣的是，在许多黄杆菌目中，编码 bS21 的基因 rpsU 包含一个扩展的 SD 序列。在这项工作中，我们提供了遗传和生化证据，表明 Flavobacterium johnsoniae 中 bS21 的合成是通过一种特定缺乏 bS21 的核糖体亚群进行自我调节的。细胞中 bS21 的突变或缺失会增加具有强 SD 序列的报告基因（如 rpsU'-gfp）的翻译，但对其他报告基因没有影响。缺乏 bS21（或其 C 末端区域）的纯化核糖体在 rpsU mRNA 上的起始率更高，而在其他（无 SD）mRNA 上的起始率更低，与对照核糖体相比。自我调节的机制依赖于 mRNA 和 16S rRNA 之间的广泛配对，以及异常强的 SD 序列，其预测配对自由能<-13 kcal/mol，这是整个拟杆菌门中 rpsU 的特征。这项工作揭示了细菌中专门化核糖体的一个明显例子。
