title: Mechanistic insights into the alternative translation termination by ArfA and RF2.
abstract: During cellular translation of messenger RNAs by ribosomes, the translation apparatus sometimes pauses or stalls at the elongation and termination steps. With the exception of programmed stalling, which is usually used by cells for regulatory purposes, ribosomes stalled on mRNAs need to be terminated and recycled to maintain adequate translation capacity. Much ribosome stalling originates in aberrant mRNAs that lack a stop codon. Transcriptional errors, misprocessing of primary transcripts, and undesired mRNA cleavage all contribute to the formation of non-stop mRNAs. Ribosomes stalled at the 3' end of non-stop mRNAs do not undergo normal termination owing to the lack of specific stop-codon recognition by canonical peptide release factors at the A-site decoding centre. In bacteria, the transfer-messenger RNA (tmRNA)-SmpB-mediated trans-translation rescue system reroutes stalled ribosomes to the normal elongation cycle and translation termination. Two additional rescue systems, ArfA-RF2 (refs 13, 14, 15, 16) and ArfB (formerly known as YaeJ), are also present in many bacterial species, but their mechanisms are not fully understood. Here, using cryo-electron microscopy, we characterize the structure of the Escherichia coli 70S ribosome bound with ArfA, the release factor RF2, a short non-stop mRNA and a cognate P-site tRNA. The C-terminal loop of ArfA occupies the mRNA entry channel on the 30S subunit, whereas its N terminus is sandwiched between the decoding centre and the switch loop of RF2, leading to marked conformational changes in both the decoding centre and RF2. Despite the distinct conformation of RF2, its conserved catalytic GGQ motif is precisely positioned next to the CCA-end of the P-site tRNA. These data illustrate a stop-codon surrogate mechanism for ArfA in facilitating the termination of non-stop ribosomal complexes by RF2.
authors.affiliation: Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Chengying
authors.initials: C
authors.lastname: Ma
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kurita
authors.affiliation: Peking-Tsinghua Center for Life Sciences, School of Life Sciences, Peking University, Beijing 100871, China.
authors.forename: Ningning
authors.initials: N
authors.lastname: Li
authors.affiliation: Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan.
authors.forename: Hyouta
authors.initials: H
authors.lastname: Himeno
authors.affiliation: Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
mesh_terms: D018388:Codon, Terminator; D020285:Cryoelectron Microscopy; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008958:Models, Molecular; D010443:Peptide Chain Termination, Translational; D010454:Peptide Termination Factors / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D011487:Protein Conformation; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D054680:Ribosome Subunits, Small, Bacterial / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: C556014:ArfA protein, E coli; D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C489006:prfB protein, E coli