Anatomy of footprint extension in ribosome profiling reveals translational landscape mediated by S1 protein in bacteria
Anatomy of footprint extension in ribosome profiling reveals translational landscape mediated by S1 protein in bacteria
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DOI：
10.21203/rs.3.rs-41219/v1
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发表时间：
2020-07
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影响因子：
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通讯作者：
Tomoya Fujita;T. Yokoyama;M. Shirouzu;H. Taguchi;Takuhiro Ito;Shintaro Iwasaki
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文献类型：
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作者：
Tomoya Fujita;T. Yokoyama;M. Shirouzu;H. Taguchi;Takuhiro Ito;Shintaro Iwasaki
核糖体分析-核糖体结合mRNA的RNA酶足迹法-是一种独特而有效的方法，应用于广泛的生物体来调查核糖体沿沿着mRNA的遍历。与真核生物相比，细菌核糖体足迹显示出广泛的大小范围，反映了核糖体的不同状态。然而，起源仍不清楚。在这里，我们发现核糖体的旋转状态和分子内RNA双链体各自在5′端延伸细菌核糖体足迹。结合延伸抑制剂，冷冻电子显微镜和核糖体分析，我们证明了核糖体的旋转状态导致长足迹。沿着亚基旋转，核糖体蛋白S1 -一种30 S亚基RNA结合蛋白-在空间上保护核糖体5′端的mRNA免受RNA酶消化，并促进核糖体的延伸。此外，我们发现，核糖体停滞在ycbZ mRNA产生延长的足迹，因为它们独特的RNA二级结构接近核糖体。通过足迹延伸的研究，我们的研究结果揭示了S1介导的翻译延伸的稳定性，并提供了核糖体分析方法来探测细菌核糖体的构象多样性。
Ribosome profiling — RNase footprinting of ribosome-bound mRNA — has been a unique and powerful method, applied to widespread organisms to survey ribosome traversal along mRNAs. In contrast to eukaryotes, bacterial ribosome footprints show a broad range of sizes, reflecting the differential states of ribosomes. However, the origin remains unclear. Here, we show that rotated state of ribosomes and intramolecular RNA duplexes each extend bacterial ribosome footprints at the 5′ end. Combining elongation inhibitors, cryo-electron microscopy, and ribosome profiling, we demonstrated that the rotated state of ribosomes results in long footprints. Along the subunit rotation, ribosomal protein S1 — a 30S-subunit RNA-binding protein — sterically protects mRNA at the 5′ end of the ribosome from RNase digestion and facilitates elongation of the ribosome. Moreover, we found that ribosomes stalled on ycbZ mRNA generate prolonged footprints because of their unique RNA secondary structure proximal to ribosomes. Through the studies of footprint extension, our results revealed S1-mediated stabilization of translation elongation and provide ribosome profiling approach to probe the conformational diversity of ribosomes in bacteria.