Alternative Fates of Paused Ribosomes during Translation Termination

Abstract

The bacterial tmRNA⅐SmpB system facilitates recycling of stalled translational complexes in a process termed "ribosome rescue." During ribosome rescue, the nascent chain is tagged with the tmRNA-encoded ssrA peptide, which targets the tagged polypeptide for degradation. Translational pausing also induces a variety of recoding events such as frameshifts, ribosome hops, and stop codon readthrough. To examine the interplay between recoding and ribosome rescue, we determined the various fates of ribosomes that pause during translation termination. We expressed a model protein containing the C-terminal Asp-Pro nascent peptide motif (which interferes with translation termination) and quantified the protein chains produced by recoding and ssrA-peptide tagging. The nature and extent of translational recoding depended upon the codon for the C-terminal Pro residue, with CCU and CCC promoting efficient ؉1 frameshifting. In contrast, ssrA-peptide tagging was unaffected by C-terminal Pro coding. Moreover, ؉1 frameshifting was not suppressed by tmRNA⅐SmpB activity, suggesting that recoding and ribosome rescue are not competing events. However, cells lacking ribosomal protein L9 (⌬L9) exhibited a significant increase in recoding and a concomitant decrease in ssrA-peptide tagging. Pulse-chase analysis revealed that pre-termination ribosomes turn over more rapidly in ⌬L9 cells, suggesting that increased recoding alleviates the translational arrest. Together, these results indicate that tmRNA⅐SmpB does not suppress transient ribosome pauses, but responds to prolonged translational arrest.

Asc1, homolog of human RACK1, prevents frameshifting in yeast by ribosomes stalled at CGA codon repeats

“…Ribosomes stall due to a variety of translational impediments, including structures in the mRNA (Doma and Parker 2006), the absence of stop codons van Hoof et al 2002), the presence of inhibitory amino acid combinations (Gutierrez et al 2013;Woolstenhulme et al 2013), or the presence of inhibitory codon combinations (Letzring et al 2010;Seidman et al 2011). Such sequences may substantially impact downstream translation.…”

“…In bacteria, such as Escherichia coli, stalled ribosomes are sometimes rescued by the trans-acting tmRNA·SmpB system (Moore and Sauer 2007), but may also undergo translational recoding, in particular +1 frameshifting (Seidman et al 2011). Errors in which ribosomes switch reading frames generally occur at extremely low rates, 3 × 10 −5 (Kurland 1992), and are almost certain to result in a nonfunctional polypeptide.…”

“…Since stalled ribosomes in Escherichia coli sometimes frameshift (Seidman et al 2011), we considered that Asc1 might not only restrict continued translation by ribosomes paused at internal CGA codon repeats (Letzring et al 2013), but might also prevent recoding events. We provide evidence here that, in the absence of Asc1 protein, ribosomes stalled at an internal CGA codon repeat undergo substantial frameshifting.…”

Crippling the Essential GTPase Der Causes Dependence on Ribosomal Protein L9

“…Subsequent studies demonstrated that L9 also influences frameshifting at codon repeats and stop codons (12,(14)(15)(16). More recently, L9 deletion strains were shown to read through stop codons more frequently (out of frame) and encounter the 3= ends of their engaged mRNAs, which then invokes ribosome rescue systems (8). Thus, at least one role for L9 is in maintaining translation fidelity, but nothing is known about the mechanism for this activity.…”

“…Interestingly, some very highly conserved ribosomal proteins can be deleted from bacteria without inducing appreciable growth phenotypes, which obfuscates determination of their molecular functions (2,6). The bacterium-specific ribosomal protein L9 is an example of this nonessential class: it possesses a conserved secondary and tertiary architecture and contains several invariant amino acids, yet deletion strains grow well (7)(8)(9). From the perspective that all highly conserved factors serve as windows to important cellular processes, we reasoned that a deeper understanding of L9 would help connect this enigmatic ribosomal protein to basic bacterial physiology.…”