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.

Mechanism of ribosome rescue by ArfA and RF2

“…After submission of this manuscript, several other structures of 70S•ArfA•RF2 complexes with RF2 in the extended conformation were reported (James et al, 2016; Huter et al, 2017; Ma et al, 2017; Zeng et al, 2017). Structure I, however, was not found in these studies.…”

“…Following the submission of our manuscript, several groups reported 70S•ArfA•RF2 cryo-EM structures (James et al, 2016; Huter et al, 2017; Ma et al, 2017; Zeng et al, 2017), but each dataset contained a single ArfA•RF2 conformation (similar to our Structure I and Structure II), unlike our dataset, which contained both ArfA•RF2 states. We hypothesize that this difference may be due to several factors.…”

The structural basis for release factor activation during translation termination revealed by time-resolved cryogenic electron microscopy

“…Further cryo-EM work showed that ribosome-bound RF1 and RF2 have extended structures 2,3 , facilitating coordinated codon recognition in DC and ester bond hydrolysis in PTC. Subsequent high-resolution X-ray crystal 7,16,17,18,19,20,21,22 and cryo-EM 5,6,23,24,25,26 structures of RF-bound 70S ribosomes allowed the modeling of stop-codon recognition by RF1, RF2 27 , eRF1 28 and GGQ-induction of ester bond hydrolysis 28 .…”

Self-Referential Encoding on Modules of Anticodon Pairs—Roots of the Biological Flow System

“…There are many other instances of reduction of the tRNA set, most appealing the generally forbidden 5’A anticodons. This rationale would be part of the RNA World proposition [57,58,59], indicating that RNAs started the construction of the biosystem and at some point developed the proteins as functional helpers for structures and catalysis, and developed the DNA as more stable genomic helpers, thereafter receding to the job of intermediates between those. These propositions seem to be now being made more flexible in favor of the coevolutionary panorama [16], that is, some kind of an RNP World preceding the present cellular DNP World, which is in entire consistency with the proposal of the Self-Referential Model (SRM), as sketched in Figure 2.…”