Implicated in persistence and stress response pathways in bacteria, RelE shuts down protein synthesis by cleaving mRNA within the ribosomal A site. Structural and biochemical studies have shown that ...RelE cuts with some sequence specificity, which we further characterize here, and that it shows no activity outside the context of the ribosome. We obtained a global view of the effect of RelE on translation by ribosome profiling, observing that ribosomes accumulate on the 5'-end of genes through dynamic cycles of mRNA cleavage, ribosome rescue and initiation. Moreover, the addition of purified RelE to cell lysates shows promise as a method for generating ribosome footprints. In bacteria, profiling studies have suffered from relatively low resolution and have yielded no information on reading frame due to problems inherent to MNase digestion, the method used to degrade unprotected regions of mRNA. In contrast, we find that RelE yields precise 3'-ends that for the first time reveal reading frame in bacteria. Given that RelE has been shown to function in all three domains of life, RelE has potential to improve reading frame and shed light on A-site occupancy in ribosome profiling experiments more broadly.
Ribosome rescue pathways recycle stalled ribosomes and target problematic mRNAs and aborted proteins for degradation
. In bacteria, it remains unclear how rescue pathways distinguish ribosomes ...stalled in the middle of a transcript from actively translating ribosomes
. Here, using a genetic screen in Escherichia coli, we discovered a new rescue factor that has endonuclease activity. SmrB cleaves mRNAs upstream of stalled ribosomes, allowing the ribosome rescue factor tmRNA (which acts on truncated mRNAs
) to rescue upstream ribosomes. SmrB is recruited to ribosomes and is activated by collisions. Cryo-electron microscopy structures of collided disomes from E. coli and Bacillus subtilis show distinct and conserved arrangements of individual ribosomes and the composite SmrB-binding site. These findings reveal the underlying mechanisms by which ribosome collisions trigger ribosome rescue in bacteria.
We used ribosome profiling to characterize the biological role of ribosome recycling factor (RRF) in
. As expected, RRF depletion leads to enrichment of post-termination 70S complexes in 3'-UTRs. We ...also observe that elongating ribosomes are unable to complete translation because they are blocked by non-recycled ribosomes at stop codons. Previous studies have suggested a role for recycling in translational coupling within operons; if a ribosome remains bound to an mRNA after termination, it may re-initiate downstream. We found, however, that RRF depletion did not significantly affect coupling efficiency in reporter assays or in ribosome density genome-wide. These findings argue that re-initiation is not a major mechanism of translational coupling in
. Finally, RRF depletion has dramatic effects on the activity of ribosome rescue factors tmRNA and ArfA. Our results provide a global view of the effects of the loss of ribosome recycling on protein synthesis in
Culviner and Laub (2018) use RNA-seq and ribosome profiling to determine how MazF inhibits translation in E. coli. Challenging an earlier model, they argue that MazF cleaves mRNA and blocks ribosome ...biogenesis but does not generate specialized ribosomes that preferentially translate leaderless transcripts.
Culviner and Laub (2018) use RNA-seq and ribosome profiling to determine how MazF inhibits translation in E. coli. Challenging an earlier model, they argue that MazF cleaves mRNA and blocks ribosome biogenesis but does not generate specialized ribosomes that preferentially translate leaderless transcripts.
Translation elongation is essential for maintaining cellular proteostasis, and alterations in the translational landscape are associated with a range of diseases. Ribosome profiling allows detailed ...measurements of translation at the genome scale. However, it remains unclear how to disentangle biological variations from technical artifacts in these data and identify sequence determinants of translation dysregulation. Here we present Riboformer, a deep learning-based framework for modeling context-dependent changes in translation dynamics. Riboformer leverages the transformer architecture to accurately predict ribosome densities at codon resolution. When trained on an unbiased dataset, Riboformer corrects experimental artifacts in previously unseen datasets, which reveals subtle differences in synonymous codon translation and uncovers a bottleneck in translation elongation. Further, we show that Riboformer can be combined with in silico mutagenesis to identify sequence motifs that contribute to ribosome stalling across various biological contexts, including aging and viral infection. Our tool offers a context-aware and interpretable approach for standardizing ribosome profiling datasets and elucidating the regulatory basis of translation kinetics.
Small proteins encoded by open reading frames (ORFs) shorter than 50 codons (small ORFs sORFs) are often overlooked by annotation engines and are difficult to characterize using traditional ...biochemical techniques. Ribosome profiling has tremendous potential to empirically improve the annotations of prokaryotic genomes. Recent improvements in ribosome profiling methods for bacterial model organisms have revealed many new sORFs in well-characterized genomes. Antibiotics that trap ribosomes just after initiation have played a key role in these developments by allowing the unambiguous identification of the start codons (and, hence, the reading frame) for novel ORFs. Here, we describe these new methods and highlight critical controls and considerations for adapting ribosome profiling to different prokaryotic species.
Ribosomes trapped on mRNAs during protein synthesis need to be rescued for the cell to survive. The most ubiquitous bacterial ribosome rescue pathway is trans-translation mediated by tmRNA and SmpB. ...Genetic inactivation of trans-translation can be lethal, unless ribosomes are rescued by ArfA or ArfB alternative rescue factors or the ribosome-associated quality control (RQC) system, which in Bacillus subtilis involves MutS2, RqcH, RqcP and Pth. Using transposon sequencing in a trans-translation-incompetent B. subtilis strain we identify a poorly characterized S4-domain-containing protein YlmH as a novel potential RQC factor. Cryo-EM structures reveal that YlmH binds peptidyl-tRNA-50S complexes in a position analogous to that of S4-domain-containing protein RqcP, and that, similarly to RqcP, YlmH can co-habit with RqcH. Consistently, we show that YlmH can assume the role of RqcP in RQC by facilitating the addition of poly-alanine tails to truncated nascent polypeptides. While in B. subtilis the function of YlmH is redundant with RqcP, our taxonomic analysis reveals that in multiple bacterial phyla RqcP is absent, while YlmH and RqcH are present, suggesting that in these species YlmH plays a central role in the RQC.
The universally conserved protein elongation factor P (EF-P) facilitates translation at amino acids that form peptide bonds with low efficiency, particularly polyproline tracts. Despite its wide ...conservation, it is not essential in most bacteria and its physiological role remains unclear. Here, we show that EF-P affects the process of sporulation initiation in the bacterium Bacillus subtilis. We observe that the lack of EF-P delays expression of sporulation-specific genes. Using ribosome profiling, we observe that expression of
, encoding a transcription factor that functions as the master regulator of sporulation, is lower in a Δ
strain than the wild type. Ectopic expression of Spo0A rescues the sporulation initiation phenotype, indicating that reduced
expression explains the sporulation defect in Δ
cells. Since Spo0A is the earliest sporulation transcription factor, these data suggest that sporulation initiation can be delayed when protein synthesis is impaired.
Elongation factor P (EF-P) is a universally conserved translation factor that prevents ribosome stalling at amino acids that form peptide bonds with low efficiency, particularly polyproline tracts. Phenotypes associated with EF-P deletion are pleiotropic, and the mechanistic basis underlying many of these phenotypes is unclear. Here, we show that the absence of EF-P affects the ability of B. subtilis to initiate sporulation by preventing normal expression of Spo0A, the key transcriptional regulator of this process. These data illustrate a mechanism that accounts for the sporulation delay and further suggest that cells are capable of sensing translation stress before committing to sporulation.
Nascent peptides that block protein synthesis in bacteria Woolstenhulme, Christopher J; Parajuli, Shankar; Healey, David W ...
Proceedings of the National Academy of Sciences - PNAS,
03/2013, Letnik:
110, Številka:
10
Journal Article
Recenzirano
Odprti dostop
Although the ribosome is a very general catalyst, it cannot synthesize all protein sequences equally well. For example, ribosomes stall on the secretion monitor (SecM) leader peptide to regulate ...expression of a downstream gene. Using a genetic selection in Escherichia coli , we identified additional nascent peptide motifs that stall ribosomes. Kinetic studies show that some nascent peptides dramatically inhibit rates of peptide release by release factors. We find that residues upstream of the minimal stalling motif can either enhance or suppress this effect. In other stalling motifs, peptidyl transfer to certain aminoacyl-tRNAs is inhibited. In particular, three consecutive Pro codons pose a challenge for elongating ribosomes. The translation factor elongation factor P, which alleviates pausing at polyproline sequences, has little or no effect on other stalling peptides. The motifs that we identified are underrepresented in bacterial proteomes and show evidence of stalling on endogenous E. coli proteins.
Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In
E. coli
, these pathways are triggered by ribosome collisions through the ...recruitment of SmrB, a nuclease that cleaves the mRNA. In
B. subtilis
, the related protein MutS2 was recently implicated in ribosome rescue. Here we show that MutS2 is recruited to collisions by its SMR and KOW domains, and we reveal the interaction of these domains with collided ribosomes by cryo-EM. Using a combination of in vivo and in vitro approaches, we show that MutS2 uses its ABC ATPase activity to split ribosomes, targeting the nascent peptide for degradation through the ribosome quality control pathway. However, unlike SmrB, which cleaves mRNA in
E. coli
, we see no evidence that MutS2 mediates mRNA cleavage or promotes ribosome rescue by tmRNA. These findings clarify the biochemical and cellular roles of MutS2 in ribosome rescue in
B. subtilis
and raise questions about how these pathways function differently in diverse bacteria.
Synopsis
Ribosome stalling in the middle of a translated mRNA leads to collisions and triggers quality control pathways. Using structural and biochemical approaches, this study reveals how MutS2 detects and resolves ribosome collisions in
Bacillus subtilis
.
MutS2 selectively binds collided ribosomes through interactions with its KOW and SMR domains.
Unlike its
E. coli
homolog SmrB,
B. subtlis
MutS2 does not cleave mRNA on collided ribosomes.
MutS2 splits stalled ribosomes into subunits using its ATPase activity.
After splitting, the nascent peptide trapped on the 50 S subunit is targeted for degradation by RqcH.
Structural and biochemical studies show how the
B. subtilis
MutS2 protein separates collided ribosomes, revealing striking differences to the actions of its
E. coli
homolog SmrB.
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