Author: ["Paul Huter","Claudia Müller","Bertrand Beckert","Stefan Arenz","Otto Berninghausen","Roland Beckmann","Daniel N. Wilson"]
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Abstract
The structure of the bacterial ribosome stalled on a truncated mRNA in complex with ArfA and the release factor RF2 is presented, revealing how ArfA recruits RF2 to the ribosome and induces conformational changes within RF2 to enable translation termination in the absence of a stop codon. When ribosomes stall on messenger RNAs, translation must be terminated so that the ribosome can be recycled. One trigger for stalling is the lack of a stop codon. Bacteria have multiple rescue systems, one of which uses the ArfA protein. The groups of Daniel Wilson, Ning Gao and Hong Jin have independently solved the structure of the bacterial ribosome in complex with ArfA, the release factor RF2, an mRNA that mimics the non-stop situation, and, in the Gao work, a P-site transfer RNA. They find that the C terminus of ArfA occupies the mRNA entry channel, while the N terminus recruits RF2 and stabilizes an active conformation in the release factor, even in the absence of a stop codon. In bacteria, ribosomes stalled on truncated mRNAs that lack a stop codon are rescued by the transfer-messenger RNA (tmRNA), alternative rescue factor A (ArfA) or ArfB systems1. Although tmRNA–ribosome and ArfB–ribosome structures have been determined2,3,4,5,6,7, how ArfA recognizes the presence of truncated mRNAs and recruits the canonical termination release factor RF2 to rescue the stalled ribosomes is unclear. Here we present a cryo-electron microscopy reconstruction of the Escherichia coli 70S ribosome stalled on a truncated mRNA in the presence of ArfA and RF2. The structure shows that the C terminus of ArfA binds within the mRNA entry channel on the small ribosomal subunit, and explains how ArfA distinguishes between ribosomes that bear truncated or full-length mRNAs. The N terminus of ArfA establishes several interactions with the decoding domain of RF2, and this finding illustrates how ArfA recruits RF2 to the stalled ribosome. Furthermore, ArfA is shown to stabilize a unique conformation of the switch loop of RF2, which mimics the canonical translation termination state by directing the catalytically important GGQ motif within domain 3 of RF2 towards the peptidyl-transferase centre of the ribosome. Thus, our structure reveals not only how ArfA recruits RF2 to the ribosome but also how it promotes an active conformation of RF2 to enable translation termination in the absence of a stop codon.Cite this article
Huter, P., Müller, C., Beckert, B. et al. Structural basis for ArfA–RF2-mediated translation termination on mRNAs lacking stop codons. Nature 541, 546–549 (2017). https://doi.org/10.1038/nature20821 