Translational termination without a stop codon

Abstract

Ribosomes stall when they encounter the end of mRNA without an in-frame stop codon. In bacteria, these nonstop complexes can be rescued by alternative ribosome-rescue factor A (ArfA). Here, using electron cryomicroscopy, we have determined structures of ArfA bound to the ribosome with 3′-truncated mRNA to resolutions from 3.0 to 3.4 Å. ArfA binds within the ribosomal mRNA channel and substitutes for the absent stop codon in the A site by specifically recruiting release factor 2 (RF2), initially in a compact pre-accommodated state. A similar conformation of RF2 may occur on stop codons, suggesting a general mechanism for releasefactor-mediated translational termination in which a conformational switch leads to peptide release only when the appropriate signal is present in the A site.In bacteria, 2-4% of mRNA transcripts lack an in-frame stop codon due to faulty transcription or nucleolytic cleavage (1). When translated, the inability to recruit release factors causes ribosomes to stall at the 3′ end of these nonstop transcripts. Translating ribosomes also stall at the 3′ end of intact transcripts when a stop codon is either read through or bypassed by translational frameshifting (2, 3). As the accumulation of stalled ribosomes is potentially lethal (4), bacteria have evolved various mechanisms to rescue these complexes (2, 3). Trans-translation is the primary rescue mechanism, present in nearly all sequenced bacterial species, and redirects ribosomes to resume translation on transfermessenger RNA (tmRNA). The reading frame of tmRNA encodes a degradation signal with a stop codon, which results in both recycling of the stalled ribosome and proteolysis of the aberrant polypeptide. Trans-translation is a promising target for antibiotic development (4); however, any therapeutic approach would need to circumvent the back-up mechanisms of alternative ribosome-rescue factors A (ArfA) and B (ArfB) which can allow some species of bacteria to survive in the absence of a functional trans-translation system (2, 3).ArfA in particular acts as a fail-safe for trans-translation in many bacterial species (5). In Escherichia coli, ArfA can support continued growth in the absence of trans-translation with few phenotypic consequences (5, 6). Under normal conditions, the arfA transcript is cleaved by RNase III to produce a nonstop mRNA substrate for trans-translation, resulting in the truncated ArfA protein being tagged for degradation (7-9), although a small constitutively expressed population of full-length ArfA may result from the translation of uncleaved arfA transcripts (7). When trans-translation is impaired or overwhelmed, full-length ArfA may rescue the synthesis of its truncated form. ArfA relieves stalled ribosomes through a Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts mechanism that requires peptidyl-tRNA hydrolysis by release factor 2 (RF2) but not the paralogous release factor 1 (RF1) (10,11). This is in contrast to ArfB, which shares homology with the catalytic domains of these rele...

Mechanism of ribosome rescue by ArfA and RF2

“…Structure I, however, was not found in these studies. A compact conformation of RF2, similar to that in Structure I, was observed in the presence of a heterologous ArfA•RF2 system or the ArfA A18T mutant (James et al, 2016). The observation of both the compact and extended RF2 conformations in a single dataset in our work could be the result of different conditions of complex preparation and/or data processing and classification (see Materials and methods).…”

“…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.…”

Extensive Ribosome and RF2 Rearrangements during Translation Termination

“…The short-lived transition from compact to open conformation(s) lasts tens of microseconds (Fu et al, 2018), and compact RF2 conformations were not captured in our sample. Compact RF2 was also visualized by cryo-EM on the 70S ribosome in the presence of truncated mRNA and alternative rescue factor A (ArfA) James et al, 2016), suggesting a conserved pathway for initial binding of RFs to non-rotated ribosomes. Together with these recent studies, our work provides an expanded structural view of the termination course -from initial codon recognition to peptide release and RF2 dissociation (Figure 4).…”

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

“…Termination may be achieved through a variety of ways, including the traditional absence of X anticodons, but it may occur even in the absence of the X codons, where it may rely upon directions given by other features of the mRNA 3' end [58,59].…”