Ribosomes translate genetic information into polypeptides in several basic steps: initiation, elongation, termination and recycling. When ribosomes are arrested during elongation or termination, the ...cell's capacity for protein synthesis is reduced. There are numerous quality control systems in place to distinguish between paused ribosomes that need some extra input to proceed and terminally stalled ribosomes that need to be rescued. Here, we discuss similarities and differences in the systems for resolution of pauses and rescue of arrested ribosomes in bacteria and eukaryotes, and how ribosome profiling has transformed our ability to decipher these molecular events.
This article is part of the themed issue ‘Perspectives on the ribosome’.
Translation factor eIF5A, containing the unique amino acid hypusine, was originally shown to stimulate Met-puromycin synthesis, a model assay for peptide bond formation. More recently, eIF5A was ...shown to promote translation elongation; however, its precise requirement in protein synthesis remains elusive. We use in vivo assays in yeast and in vitro reconstituted translation assays to reveal a specific requirement for eIF5A to promote peptide bond formation between consecutive Pro residues. Addition of eIF5A relieves ribosomal stalling during translation of three consecutive Pro residues in vitro, and loss of eIF5A function impairs translation of polyproline-containing proteins in vivo. Hydroxyl radical probing experiments localized eIF5A near the E site of the ribosome with its hypusine residue adjacent to the acceptor stem of the P site tRNA. Thus, eIF5A, like its bacterial ortholog EFP, is proposed to stimulate the peptidyl transferase activity of the ribosome and facilitate the reactivity of poor substrates like Pro.
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•eIF5A mutant is defective for translation of polyproline sequences in vivo•eIF5A alleviates ribosomal stalling on polyproline sequences in vitro•Hypusine modification of eIF5A stimulates polyproline synthesis in vitro•Ribosome binding of eIF5A places hypusine adjacent to acceptor stem of P site tRNA
Ribosome profiling is a powerful method for globally assessing the activity of ribosomes in a cell. Despite its application in many organisms, ribosome profiling studies in bacteria have struggled to ...obtain the resolution necessary to precisely define translational pauses. Here, we report improvements that yield much higher resolution in E. coli profiling data, enabling us to more accurately assess ribosome pausing and refine earlier studies of the impact of polyproline motifs on elongation. We comprehensively characterize pausing at proline-rich motifs in the absence of elongation factor EFP. We find that only a small fraction of genes with strong pausing motifs have reduced ribosome density downstream, and we identify features that explain this phenomenon. These features allow us to predict which proteins likely have reduced output in the efp-knockout strain.
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•The 3′ end of footprints best defines occupancy in bacterial ribosome profiling•Though pausing at Pro-Pro motifs is widespread, context determines the intensity•Strong pauses near the 5′ end of genes reduce downstream ribosome density
By improving the resolution of ribosome profiling, Woolstenhulme et al. provide a clear view of translational pauses in E. coli. They characterize pausing at polyproline motifs in bacteria lacking EFP, providing insight into the breadth and intensity of pausing and how pausing impacts protein output.
In eukaryotes, ribosome profiling provides insight into the mechanism of protein synthesis at the codon level. In bacteria, however, the method has been more problematic and no consensus has emerged ...for how to best prepare profiling samples. Here, we identify the sources of these problems and describe new solutions for arresting translation and harvesting cells in order to overcome them. These improvements remove confounding artifacts and improve the resolution to allow analyses of ribosome behavior at the codon level. With a clearer view of the translational landscape in vivo, we observe that filtering cultures leads to translational pauses at serine and glycine codons through the reduction of tRNA aminoacylation levels. This observation illustrates how bacterial ribosome profiling studies can yield insight into the mechanism of protein synthesis at the codon level and how these mechanisms are regulated in response to changes in the physiology of the cell.
The rate of protein synthesis varies according to the mRNA sequence in ways that affect gene expression. Global analysis of translational pausing is now possible with ribosome profiling. Here, we ...revisit an earlier report that Shine-Dalgarno sequences are the major determinant of translational pausing in bacteria. Using refinements in the profiling method as well as biochemical assays, we find that SD motifs have little (if any) effect on elongation rates. We argue that earlier evidence of pausing arose from two factors. First, in previous analyses, pauses at Gly codons were difficult to distinguish from pauses at SD motifs. Second, and more importantly, the initial study preferentially isolated long ribosome-protected mRNA fragments that are enriched in SD motifs. These findings clarify the landscape of translational pausing in bacteria as observed by ribosome profiling.
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•Ribosome footprints containing Shine-Dalgarno motifs are longer than other footprints•An earlier study reporting pauses at SD motifs preferentially isolated long footprints•Pauses at SD motifs within open reading frames are not observed in vitro
An earlier study reported that Shine-Dalgarno motifs are the major cause of translational pausing in bacteria. Using ribosome profiling and biochemical assays, Mohammad et al. demonstrate that SD motifs have little or no effect on elongation rates and pinpoint the source of the reported SD pauses.
High-throughput methods, such as ribosome profiling, have revealed the complexity of translation regulation in Bacteria and Eukarya with large-scale effects on cellular functions. In contrast, the ...translational landscape in Archaea remains mostly unexplored. Here, we developed ribosome profiling in a model archaeon, Haloferax volcanii, elucidating, for the first time, the translational landscape of a representative of the third domain of life. We determined the ribosome footprint of H. volcanii to be comparable in size to that of the Eukarya. We linked footprint lengths to initiating and elongating states of the ribosome on leadered transcripts, operons, and on leaderless transcripts, the latter representing 70% of H. volcanii transcriptome. We manipulated ribosome activity with translation inhibitors to reveal ribosome pausing at specific codons. Lastly, we found that the drug harringtonine arrested ribosomes at initiation sites in this archaeon. This drug treatment allowed us to confirm known translation initiation sites and also reveal putative novel initiation sites in intergenic regions and within genes. Ribosome profiling revealed an uncharacterized complexity of translation in this archaeon with bacteria-like, eukarya-like, and potentially novel translation mechanisms. These mechanisms are likely to be functionally essential and to contribute to an expanded proteome with regulatory roles in gene expression.
The eukaryotic translation factor eIF5A, originally identified as an initiation factor, was later shown to promote translation elongation of iterated proline sequences. Using a combination of ...ribosome profiling and in vitro biochemistry, we report a much broader role for eIF5A in elongation and uncover a critical function for eIF5A in termination. Ribosome profiling of an eIF5A-depleted strain reveals a global elongation defect, with abundant ribosomes stalling at many sequences, not limited to proline stretches. Our data also show ribosome accumulation at stop codons and in the 3′ UTR, suggesting a global defect in termination in the absence of eIF5A. Using an in vitro reconstituted translation system, we find that eIF5A strongly promotes the translation of the stalling sequences identified by profiling and increases the rate of peptidyl-tRNA hydrolysis more than 17-fold. We conclude that eIF5A functions broadly in elongation and termination, rationalizing its high cellular abundance and essential nature.
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•Depletion of eIF5A leads to global translation elongation and termination defects•eIF5A alleviates stalling on many motifs besides polyproline tracts•eIF5A stimulates eRF1-mediated peptidyl-tRNA hydrolysis in translation termination
In this manuscript, Schuller et al. characterize the activity of the translation factor eIF5A. They show that eIF5A accelerates peptidyl transfer at most, if not all, sequences and promotes peptide release. These expanded roles for eIF5A help to explain its essential nature and high abundance in eukaryotes.
Small proteins consisting of 50 or fewer amino acids have been identified as regulators of larger proteins in bacteria and eukaryotes. Despite the importance of these molecules, the total number of ...small proteins remains unknown because conventional annotation pipelines usually exclude small open reading frames (smORFs). We previously identified several dozen small proteins in the model organism
using theoretical bioinformatic approaches based on sequence conservation and matches to canonical ribosome binding sites. Here, we present an empirical approach for discovering new proteins, taking advantage of recent advances in ribosome profiling in which antibiotics are used to trap newly initiated 70S ribosomes at start codons. This approach led to the identification of many novel initiation sites in intergenic regions in
We tagged 41 smORFs on the chromosome and detected protein synthesis for all but three. Not only are the corresponding genes intergenic but they are also found antisense to other genes, in operons, and overlapping other open reading frames (ORFs), some impacting the translation of larger downstream genes. These results demonstrate the utility of this method for identifying new genes, regardless of their genomic context.
Proteins comprised of 50 or fewer amino acids have been shown to interact with and modulate the functions of larger proteins in a range of organisms. Despite the possible importance of small proteins, the true prevalence and capabilities of these regulators remain unknown as the small size of the proteins places serious limitations on their identification, purification, and characterization. Here, we present a ribosome profiling approach with stalled initiation complexes that led to the identification of 38 new small proteins.
Shine-Dalgarno (SD) motifs are thought to play an important role in translational initiation in bacteria. Paradoxically, ribosome profiling studies in
show no correlation between the strength of an ...mRNA's SD motif and how efficiently it is translated. Performing profiling on ribosomes with altered anti-Shine-Dalgarno sequences, we reveal a genome-wide correlation between SD strength and ribosome occupancy that was previously masked by other contributing factors. Using the antibiotic retapamulin to trap initiation complexes at start codons, we find that the mutant ribosomes select start sites correctly, arguing that start sites are hard-wired for initiation through the action of other mRNA features. We show that A-rich sequences upstream of start codons promote initiation. Taken together, our genome-wide study reveals that SD motifs are not necessary for ribosomes to determine where initiation occurs, though they do affect how efficiently initiation occurs.
Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still ...unknown. Here, we use a combination of in vitro biochemistry, ribosome profiling, and cryo‐EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an in vitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base‐pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo‐EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding‐incompatible conformations of mRNA in the A sites of all studied stall‐ and collision‐inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA‐induced mechanisms of translational stalling in eukaryotic ribosomes.
Synopsis
A combination of in vitro biochemistry, ribosome profiling, and cryo‐EM defines molecular events that lead to ribosome stalling at inhibitory codon combinations and poly(A) tracts, demonstrating novel mRNA‐induced mechanisms slowing translational elongation by eukaryotic ribosomes.
Inhibitory codon pairs and poly(A) tracts within mRNAs form decoding‐incompatible conformations in the decoding center of the ribosome.
Direct effects of these structures on decoding are documented by high‐resolution ribosome profiling, kinetic studies and detailed cryo‐EM structural analysis.
These data demonstrate a novel mRNA‐induced mechanism of translational stalling in eukaryotic ribosomes.
The cryo‐EM structure of poly(A)‐stalled disomes reveals a novel conformation for collided ribosomes that rationalizes translational frameshifting.
A combination of in vitro biochemistry, ribosome profiling, and cryo‐EM reveals that elongation‐slowing mRNA elements stall ribosomes in decoding‐incompatible RNA conformations in the ribosomal decoding center.
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