在噬菌体λ溶原中,λcI阻遏物由在λpRM启动子处起始的无前导序列转录物(lmRNA)编码。在缺乏核糖体蛋白uS2的rpsB突变体中翻译增强。尽管lmRNA的翻译起始在细菌中是保守的,古细菌,和真核生物,缺乏lmRNA翻译起始复合物的结构洞察力。这里,我们使用cryo-EM来解决宿主大肠杆菌突变体rpsB11的uS2缺陷70S核糖体的结构和具有λcIlmRNA和fMet-tRNAfMet的野生型70S复合物。重要的是,uS2缺陷的70S核糖体也缺乏蛋白bS21。抗Shine-Dalgarno(aSD)区域在结构上由bS21支持,因此后者的缺失导致aSD从正常的mRNA出口途径转移,缓解lmRNA的退出。监测碱基A1493和mRNA的A(+4)之间的π-堆积相互作用潜在地充当识别信号。库仑电荷流,由于缺乏uS2导致30S头部旋转增加,mRNA入口通道内的蠕动样动力学可能会促进lmRNA通过核糖体的传播。这些发现为未来研究lmRNA和mRNA的翻译机制和共同进化奠定了基础,包括转录本的确定的核糖体结合位点的出现。
In bacteriophage λ lysogens, the λcI repressor is encoded by the leaderless transcript (lmRNA) initiated at the λpRM promoter. Translation is enhanced in rpsB mutants deficient in ribosomal protein uS2. Although translation initiation of lmRNA is conserved in bacteria, archaea, and eukaryotes, structural insight of a lmRNA translation initiation complex is missing. Here, we use cryo-EM to solve the structures of the uS2-deficient 70S ribosome of host E. coli mutant rpsB11 and the wild-type 70S complex with λcI lmRNA and fMet-tRNAfMet. Importantly, the uS2-deficient 70S ribosome also lacks protein bS21. The anti-Shine-Dalgarno (aSD) region is structurally supported by bS21, so that the absence of the latter causes the aSD to divert from the normal mRNA exit pathway, easing the exit of lmRNA. A π-stacking interaction between the monitor base A1493 and A(+4) of lmRNA potentially acts as a recognition signal. Coulomb charge flow, along with peristalsis-like dynamics within the mRNA entrance channel due to the increased 30S head rotation caused by the absence of uS2, are likely to facilitate the propagation of lmRNA through the ribosome. These findings lay the groundwork for future research on the mechanism of translation and the co-evolution of lmRNA and mRNA that includes the emergence of a defined ribosome-binding site of the transcript.