RNA Therapeutics Institute, Department of Biochemistry and Molecular Pharmacology, UMass Medical School, Worcester, Massachusetts, 01605, United States.
Central European Institute of Technology, Masaryk University, Kamenice 5, Brno, 625 00, Czech Republic.
Nat Commun. 2020 Nov 3;11(1):5552. doi: 10.1038/s41467-020-19370-z.
Ribosomes stalled during translation must be rescued to replenish the pool of translation-competent ribosomal subunits. Bacterial alternative rescue factor B (ArfB) releases nascent peptides from ribosomes stalled on mRNAs truncated at the A site, allowing ribosome recycling. Prior structural work revealed that ArfB recognizes such ribosomes by inserting its C-terminal α-helix into the vacant mRNA tunnel. In this work, we report that ArfB can efficiently recognize a wider range of mRNA substrates, including longer mRNAs that extend beyond the A-site codon. Single-particle cryo-EM unveils that ArfB employs two modes of function depending on the mRNA length. ArfB acts as a monomer to accommodate a shorter mRNA in the ribosomal A site. By contrast, longer mRNAs are displaced from the mRNA tunnel by more than 20 Å and are stabilized in the intersubunit space by dimeric ArfB. Uncovering distinct modes of ArfB function resolves conflicting biochemical and structural studies, and may lead to re-examination of other ribosome rescue pathways, whose functions depend on mRNA lengths.
核糖体在翻译过程中停滞必须被拯救，以补充具有翻译能力的核糖体亚基池。细菌替代救援因子 B（ArfB）从在 A 位截断的 mRNA 上停滞的核糖体上释放新生肽，从而允许核糖体循环。先前的结构研究表明，ArfB 通过将其 C 末端α-螺旋插入空的 mRNA 隧道来识别这种核糖体。在这项工作中，我们报告说，ArfB 可以有效地识别更广泛的 mRNA 底物，包括延伸到 A 位密码子之外的更长的 mRNA。单颗粒 cryo-EM 揭示，ArfB 根据 mRNA 长度采用两种功能模式。ArfB 作为单体作用，以适应核糖体 A 位上较短的 mRNA。相比之下，较长的 mRNA 被从 mRNA 隧道中移位超过 20 Å，并由二聚体 ArfB 稳定在亚基间空间中。揭示 ArfB 功能的不同模式解决了相互矛盾的生化和结构研究，并可能导致对其他依赖于 mRNA 长度的核糖体救援途径的重新检查。
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