缺乏核糖体蛋白uS2的大肠杆菌核糖体比野生型核糖体1更有效地翻译无引线转录本没有Shine-Dalgarno序列,翻译起始发生在突变体70S复合物上而不是30S起始复合物上。核糖体使用起始密码子(AUG)和转录物的第一个碱基,包括下游盒(DB)序列,作为识别信号。我们使用cryo-EM解决了在fmet-tRNAfMet存在下与无引线mRNA(lmRNA)结合的uS2缺陷的70S核糖体和野生型70S复合物的结构。重要的是,uS2缺陷的70S核糖体也缺乏蛋白bS21。抗Shine-Dalgarno(aSD)区域由bS21支持,后者的缺乏导致aSD从正常的mRNA出口途径转移。我们鉴定了监测碱基(1493A)和AUG后的lmRNA的第一个碱基(A+4)之间的π-堆积相互作用,并可能使其稳定。库仑电荷流动和交换以及mRNA进入通道内的蠕动样动力学可能促进lmRNA通过核糖体的传播。
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 entry 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.