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Many antibiotics that bind to the ribosome inhibit translation by blocking the movement of tRNAs and mRNA or interfering with ribosome dynamics, which impairs the formation of essential translocation intermediates. Here we show how translocation inhibitors viomycin (Vio), neomycin (Neo), paromomycin (Par), kanamycin (Kan), spectinomycin (Spc), hygromycin B (HygB), and streptomycin (Str, an antibiotic that does not inhibit tRNA movement), affect principal motions of the small ribosomal subunits (SSU) during EF-G-promoted translocation. Using ensemble kinetics, we studied the SSU body domain rotation and SSU head domain swiveling in real time. We show that although antibiotics binding to the ribosome can favor a particular ribosome conformation in the absence of EF-G, their kinetic effect on the EF-G-induced transition to the rotated/swiveled state of the SSU is moderate. The antibiotics mostly inhibit backward movements of the SSU body and/or the head domains. Vio, Spc, and high concentrations of Neo completely inhibit the backward movements of the SSU body and head domain. Kan, Par, HygB, and low concentrations of Neo slow down both movements, but their sequence and coordination are retained. Finally, Str has very little effect on the backward rotation of the SSU body domain, but retards the SSU head movement. The data underscore the importance of ribosome dynamics for tRNA-mRNA translocation and provide new insights into the mechanism of antibiotic action.

Fluorescence reporter groups are important tools to study the structure and dynamics of proteins. Genetic code reprogramming allows for cotranslational incorporation of non-canonical amino acids at any desired position. However, cotranslational incorporation of bulky fluorescence reporter groups is technically challenging and usually inefficient. Here we analyze the bottlenecks for the cotranslational incorporation of NBD-, BodipyFL- and Atto520-labeled Cys-tRNACys into a model protein using a reconstituted in-vitro translation system. We show that the modified Cys-tRNACys can be rejected during decoding due to the reduced ribosome selectivity for the modified aa-tRNA and the competition with native near-cognate aminoacyl-tRNAs. Accommodation of the modified Cys-tRNACys in the A site of the ribosome is also impaired, but can be rescued by one or several Gly residues at the positions −1 to −4 upstream of the incorporation site. The incorporation yield depends on the steric properties of the downstream residue and decreases with the distance from the protein N-terminus to the incorporation site. In addition to the full-length translation product, we find protein fragments corresponding to the truncated N-terminal peptide and the C-terminal fragment starting with a fluorescence-labeled Cys arising from a StopGo-like event due to a defect in peptide bond formation. The results are important for understanding the reasons for inefficient cotranslational protein labeling with bulky reporter groups and for designing new approaches to improve the yield of fluorescence-labeled protein.

When reading consecutive mRNA codons, ribosomes move by exactly one triplet at a time to synthesize a correct protein. Some mRNA tracks, called slippery sequences, are prone to ribosomal frameshifting, because the same tRNA can read both 0- and –1-frame codon. Using smFRET we show that during EF-G-catalyzed translocation on slippery sequences a fraction of ribosomes spontaneously switches from rapid, accurate translation to a slow, frameshifting-prone translocation mode where the movements of peptidyl- and deacylated tRNA become uncoupled. While deacylated tRNA translocates rapidly, pept-tRNA continues to fluctuate between chimeric and posttranslocation states, which slows down the re-locking of the small ribosomal subunit head domain. After rapid release of deacylated tRNA, pept-tRNA gains unconstrained access to the –1-frame triplet, resulting in slippage followed by recruitment of the –1-frame aa-tRNA into the A site. Our data show how altered choreography of tRNA and ribosome movements reduces the translation fidelity of ribosomes translocating in a slow mode.

Cellular proteins begin to fold as they emerge from the ribosome. The folding landscape of nascent chains is not only shaped by their amino acid sequence but also by the interactions with the ribosome. Here, we combine biophysical methods with cryo-EM structure determination to show that folding of a β-barrel protein begins with formation of a dynamic α-helix inside the ribosome. As the growing peptide reaches the end of the tunnel, the N-terminal part of the nascent chain refolds to a β-hairpin structure that remains dynamic until its release from the ribosome. Contacts with the ribosome and structure of the peptidyl transferase center depend on nascent chain conformation. These results indicate that proteins may start out as α-helices inside the tunnel and switch into their native folds only as they emerge from the ribosome. Moreover, the correlation of nascent chain conformations with reorientation of key residues of the ribosomal peptidyl-transferase center suggest that protein folding could modulate ribosome activity., The work was funded by the Max Planck Society to M.V.R., the European Research Council (ERC) Advanced Investigator Grant RIBOFOLD to M.V.R. (proposal number n° 787926), and the Spanish Ministry of Economy and Competitiveness to X.A. (CTQ2015-73560-JIN) and to M.V. (PGC2018-098996-B-I00). We thank the Spanish Ministry of Science for the Severo Ochoa Excellence Accreditations to the CIC bioGUNE (SEV-2016-0644). CIISB research infrastructure project LM2018127 funded by MEYS CR is gratefully acknowledged for the financial support of the measurements at the CF Cryo-electron Microscopy and Tomography. Open Access funding enabled and organized by Projekt DEAL.

SummaryTranslational readthrough (TR) occurs when the ribosome decodes a stop codon as a sense codon, resulting in two protein isoforms synthesized from the same mRNA. TR is pervasive in eukaryotic organisms; however, its biological significance remains unclear. In this study, we quantify the TR potential of several candidate genes inDrosophila melanogasterand characterize the regulation of TR in the large Maf transcription factor Traffic jam (Tj). We used CRISPR/Cas9 generated mutant flies to show that the TR-generated Tj isoform is expressed in the nuclei of a subset of neural cells of the central nervous system and is excluded from the somatic cells of gonads, which express the short Tj isoform only. Translational control of TR is critical for preservation of neuronal integrity and maintenance of reproductive health. Fine-tuning of the gene regulatory functions of transcription factors by TR provides a new potential mechanism for cell-specific regulation of gene expression.HighlightsTj undergoes tissue-specific TR in neural cells of the central nervous system.Strict control of TR is crucial for neuroprotection and maintenance of reproductive capacity.TR selectively fine-tunes the gene regulatory functions of the transcription factor.TR in Tj links transcription and translation of tissue-specific control of gene expression.

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP–Pi form stabilizes the rotated conformation of ribosomal subunits and induces twisting of the sarcin-ricin loop of the 23 S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a large-scale rigid-body rotation of EF-G pivoting around the sarcin-ricin loop that facilitates back rotation of the ribosomal subunits and forward swiveling of the head domain of the small subunit, ultimately driving tRNA forward movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement., Movement of the ribosome along an mRNA requires the universally-conserved translocase (EF-G in bacteria) that couples GTP hydrolysis to directed movement. Here the authors use time-resolved Cryo-EM to visualize the GTPase-powered step on native translocating ribosomes and capture key translocation intermediates.

Significance Membrane proteins are inserted into the phospholipid bilayer through a lateral gate in the translocon, SecYEG in bacteria, which is expected to be closed in the resting state. Here, we use single-molecule FRET to study the translocon dynamics on timescales ranging from submilliseconds to seconds. We show that the lateral gate is highly dynamic, fluctuating through a continuum of states from open to closed. The insertase YidC facilitates the insertion of transmembrane helices by shifting the fluctuations toward more open conformations. Spontaneous fluctuations allow the gate to rapidly release newly synthesized transmembrane segments into the phospholipid bilayer during ongoing translation. The results highlight the important role of rapid spontaneous fluctuations during the key step in the biogenesis of inner-membrane proteins., During synthesis of membrane proteins, transmembrane segments (TMs) of nascent proteins emerging from the ribosome are inserted into the central pore of the translocon (SecYEG in bacteria) and access the phospholipid bilayer through the open lateral gate formed of two helices of SecY. Here we use single-molecule fluorescence resonance energy transfer to monitor lateral-gate fluctuations in SecYEG embedded in nanodiscs containing native membrane phospholipids. We find the lateral gate to be highly dynamic, sampling the whole range of conformations between open and closed even in the absence of ligands, and we suggest a statistical model-free approach to evaluate the ensemble dynamics. Lateral gate fluctuations take place on both short (submillisecond) and long (subsecond) timescales. Ribosome binding and TM insertion do not halt fluctuations but tend to increase sampling of the open state. When YidC, a constituent of the holotranslocon, is bound to SecYEG, TM insertion facilitates substantial opening of the gate, which may aid in the folding of YidC-dependent polytopic membrane proteins. Mutations in lateral gate residues showing in vivo phenotypes change the range of favored states, underscoring the biological significance of lateral gate fluctuations. The results suggest how rapid fluctuations of the lateral gate contribute to the biogenesis of inner-membrane proteins.

Elongation factor G (EF-G) is a translational GTPase that acts at several stages of protein synthesis. Its canonical function is to catalyze tRNA movement during translation elongation, but it also acts at the last step of translation to promote ribosome recycling. Moreover, EF-G has additional functions, such as helping the ribosome to maintain the mRNA reading frame or to slide over non-coding stretches of the mRNA. EF-G has an unconventional GTPase cycle that couples the energy of GTP hydrolysis to movement. EF-G facilitates movement in the GDP-Pi form. To convert the energy of hydrolysis to movement, it requires various ligands in the A site, such as a tRNA in translocation, an mRNA secondary structure element in ribosome sliding, or ribosome recycling factor in post-termination complex disassembly. The ligand defines the direction and timing of EF-G-facilitated motion. In this review, we summarize recent advances in understanding the mechanism of EF-G action as a remarkable force-generating GTPase.

During canonical translation, the ribosome moves along an mRNA from the start to the stop codon in exact steps of one codon at a time. The collinearity of the mRNA and the protein sequence is essential for the quality of the cellular proteome. Spontaneous errors in decoding or translocation are rare and result in a deficient protein. However, dedicated recoding signals in the mRNA can reprogram the ribosome to read the message in alternative ways. This review summarizes the recent advances in understanding the mechanisms of three types of recoding events: stop-codon readthrough, –1 ribosome frameshifting and translational bypassing. Recoding events provide insights into alternative modes of ribosome dynamics that are potentially applicable to other non-canonical modes of prokaryotic and eukaryotic translation.

Some proteins are expressed as a result of a ribosome frameshifting event that is facilitated by a slippery site and downstream secondary structure elements in the mRNA. This review summarizes recent progress in understanding mechanisms of –1 frameshifting in several viral genes, including IBV 1a/1b, HIV‐1 gag‐pol, and SFV 6K, and in Escherichia coli dnaX. The exact frameshifting route depends on the availability of aminoacyl‐tRNAs: the ribosome normally slips into the –1‐frame during tRNA translocation, but can also frameshift during decoding at condition when aminoacyl‐tRNA is in limited supply. Different frameshifting routes and additional slippery sites allow viruses to maintain a constant production of their key proteins. The emerging idea that tRNA pools are important for frameshifting provides new direction for developing antiviral therapies.

Protein homeostasis of bacterial cells is maintained by coordinated processes of protein production, folding, and degradation. Translational efficiency of a given mRNA depends on how often the ribosomes initiate synthesis of a new polypeptide and how quickly they read the coding sequence to produce a full-length protein. The pace of ribosomes along the mRNA is not uniform: periods of rapid synthesis are separated by pauses. Here, we summarize recent evidence on how ribosome pausing affects translational efficiency and protein folding. We discuss the factors that slow down translation elongation and affect the quality of the newly synthesized protein. Ribosome pausing emerges as important factor contributing to the regulatory programs that ensure the quality of the proteome and integrate the cellular and environmental cues into regulatory circuits of the cell.

In addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the ribosomal A and E sites, its exact mechanism of action is unclear. Here, we show that eEF3 acts at the mRNA–tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl‐tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slowdown in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo‐EM analysis of native eEF3‐ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non‐rotated ribosomal states, as well as by opening the L1 stalk to release the E‐site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle., A combination of rapid kinetics, ribosome profiling, and cryo‐EM structures reveals eEF3’s requirement during late steps of ribosomal translocation/ translation.

Nascent polypeptides begin to fold in the constrained space of the ribosomal peptide exit tunnel. Here we use force-profile analysis (FPA) and photo-induced energy-transfer fluorescence correlation spectroscopy (PET-FCS) to show how a small α-helical domain, the N-terminal domain of HemK, folds cotranslationally. Compaction starts vectorially as soon as the first α-helical segments are synthesized. As nascent chain grows, emerging helical segments dock onto each other and continue to rearrange at the vicinity of the ribosome. Inside or in the proximity of the ribosome, the nascent peptide undergoes structural fluctuations on the µs time scale. The fluctuations slow down as the domain moves away from the ribosome. Mutations that destabilize the packing of the domain’s hydrophobic core have little effect on folding within the exit tunnel, but abolish the final domain stabilization. The results show the power of FPA and PET-FCS in solving the trajectory of cotranslational protein folding and in characterizing the dynamic properties of folding intermediates.

Integral membrane proteins insert into the bacterial inner membrane co‐translationally via the translocon. Transmembrane (TM) segments of nascent proteins adopt their native topological arrangement with the N‐terminus of the first TM (TM1) oriented to the outside (type I) or the inside (type II) of the cell. Here, we study TM1 topogenesis during ongoing translation in a bacterial in vitro system, applying real‐time FRET and protease protection assays. We find that TM1 of the type I protein LepB reaches the translocon immediately upon emerging from the ribosome. In contrast, the type II protein EmrD requires a longer nascent chain before TM1 reaches the translocon and adopts its topology by looping inside the ribosomal peptide exit tunnel. Looping presumably is mediated by interactions between positive charges at the N‐terminus of TM1 and negative charges in the tunnel wall. Early TM1 inversion is abrogated by charge reversal at the N‐terminus. Kinetic analysis also shows that co‐translational membrane insertion of TM1 is intrinsically rapid and rate‐limited by translation. Thus, the ribosome has an important role in membrane protein topogenesis., FRET measurements reveal that transmembrane proteins with cytoplasmic N‐termini have their nascent peptide inverted within the ribosomal exit tunnel.

Protein folding begins co-translationally within the restricted space of the peptide exit tunnel of the ribosome. We have already shown that the N-terminal α-helical domain of the universally conserved N5-glutamine methyltransferase HemK is compacted within the exit tunnel and rearranges into the native fold upon emerging from the ribosome. However, the exact folding pathway of the domain remained unclear. Here we analyzed the rapid kinetics of translation and folding monitored by fluorescence resonance energy transfer and photoinduced electron transfer using global fitting to a model for synthesis of the 112-amino acid HemK fragment. Our results suggest that the co-translational folding trajectory of HemK starts within the tunnel and passes through four kinetically distinct folding intermediates that may represent sequential docking of helices to a growing compact core. The kinetics of the process is defined entirely by translation. The results show how analysis of ensemble kinetic data can be used to dissect complex trajectories of rapid conformational rearrangements in multicomponent systems.

The bacterial signal recognition particle (SRP) is part of the machinery that targets ribosomes synthesizing membrane proteins to membrane-embedded translocons co-translationally. Recognition of nascent membrane proteins occurs by virtue of a hydrophobic signal-anchor sequence (SAS) contained in the nascent chain, usually at the N terminus. Here we use fluorescence-based stopped-flow to monitor SRP-ribosome interactions with actively translating ribosomes while an SRP substrate is synthesized and emerges from the peptide exit tunnel. The kinetic analysis reveals that, at cellular concentrations of ribosomes and SRP, SRP rapidly binds to translating ribosomes prior to the emergence of an SAS and forms an initial complex that rapidly rearranges to a more stable engaged complex. When the growing peptide reaches a length of ∼50 amino acids and the SAS is partially exposed, SRP undergoes another conformational change which further stabilizes the complex and initiates targeting of the translating ribosome to the translocon. These results provide a reconciled view on the timing of high-affinity targeting complex formation, while emphasizing the existence of preceding SRP recruitment steps under conditions of ongoing translation.

Many antibiotics stop bacterial growth by inhibiting different steps of protein synthesis. However, no specific inhibitors of translation termination are known. Proline-rich antimicrobial peptides, a component of the antibacterial defense system of multicellular organisms, interfere with bacterial growth by inhibiting translation. Here we show that Api137, a derivative of the insect-produced antimicrobial peptide apidaecin, arrests terminating ribosomes using a unique mechanism of action. Api137 binds to the Escherichia coli ribosome and traps release factors 1 or 2 subsequent to release of the nascent polypeptide chain. A high-resolution cryo-EM structure of the ribosome complexed with release factor 1 and Api137 reveals the molecular interactions that lead to release factor trapping. Api137-mediated depletion of the cellular pool of free release factors causes the majority of ribosomes to stall at stop codons prior to polypeptide release, thereby resulting in a global shutdown of translation termination.

A hallmark of translation in human immunodeficiency virus type 1 (HIV-1) is a -1 programmed ribosome frameshifting event that produces the Gag-Pol fusion polyprotein. The constant Gag to Gag-Pol ratio is essential for the virion structure and infectivity. Here we show that the frameshifting efficiency is modulated by Leu-tRNALeu that reads the UUA codon at the mRNA slippery site. This tRNALeu isoacceptor is particularly rare in human cell lines derived from T-lymphocytes, the cells that are targeted by HIV-1. When UUA decoding is delayed, the frameshifting follows an alternative route, which maintains the Gag to Gag-Pol ratio constant. A second potential slippery site downstream of the first one is normally inefficient but can also support -1-frameshifting when altered by a compensatory resistance mutation in response to current antiviral drug therapy. Together these different regimes allow the virus to maintain a constant -1-frameshifting efficiency to ensure successful virus propagation. peerReviewed

Translational translocase pushes hyper-rotated ribosomes to slide along the noncoding mRNA gap at the cost of GTP hydrolysis., Translational bypassing is a recoding event during which ribosomes slide over a noncoding region of the messenger RNA (mRNA) to synthesize one protein from two discontinuous reading frames. Structures in the mRNA orchestrate forward movement of the ribosome, but what causes ribosomes to start sliding remains unclear. Here, we show that elongation factor G (EF-G) triggers ribosome take-off by a pseudotranslocation event using a small mRNA stem-loop as an A-site transfer RNA mimic and requires hydrolysis of about two molecules of guanosine 5′-triphosphate per nucleotide of the noncoding gap. Bypassing ribosomes adopt a hyper-rotated conformation, also observed with ribosomes stalled by the SecM sequence, suggesting common ribosome dynamics during translation stalling. Our results demonstrate a new function of EF-G in promoting ribosome sliding along the mRNA, in contrast to codon-wise ribosome movement during canonical translation, and suggest a mechanism by which ribosomes could traverse untranslated parts of mRNAs.

Mitochondrial gene expression uses a non‐universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt‐)tRNAMet mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt‐tRNAMet to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt‐tRNAMet to generate an f5C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilisation of m5C34 mt‐tRNAMet in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt‐tRNAMet function. Together, our data reveal how modifications in mt‐tRNAMet are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNAMet to recognise the different codons encoding methionine. ![][1] RNA methyltransferase NSUN3 acts specifically on mitochondrial tRNAMet, allowing different codons to be recognised by this single tRNA and offering insight on the consequence of reported disease mutations. [1]: /embed/graphic-1.gif

Translational GTPases (trGTPases) play key roles in facilitating protein synthesis on the ribosome. Despite the high degree of evolutionary conservation in the sequences of their GTP‐binding domains, the rates of GTP hydrolysis and nucleotide exchange vary broadly between different trGTPases. EF‐Tu, one of the best‐characterized model G proteins, evolved an exceptionally rapid and tightly regulated GTPase activity, which ensures rapid and accurate incorporation of amino acids into the nascent chain. Other trGTPases instead use the energy of GTP hydrolysis to promote movement or to ensure the forward commitment of translation reactions. Recent data suggest the GTPase mechanism of EF‐Tu and provide an insight in the catalysis of GTP hydrolysis by its unusual activator, the ribosome. Here we summarize these advances in understanding the functional cycle and the regulation of trGTPases, stimulated by the elucidation of their structures on the ribosome and the progress in dissecting the reaction mechanism of GTPases. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 463–475, 2016.

The elongation phase of protein synthesis defines the overall speed and fidelity of protein synthesis and affects protein folding and targeting. The mechanisms of reactions taking place during translation elongation remain important questions in understanding ribosome function. The ribosome-guided by signals in the mRNA-can recode the genetic information, resulting in alternative protein products. Co-translational protein folding and interaction of ribosomes and emerging polypeptides with associated protein biogenesis factors determine the quality and localization of proteins. In this review, we summarize recent findings on mechanisms of translation elongation in bacteria, including decoding and recoding, peptide bond formation, tRNA-mRNA translocation, co-translational protein folding, interaction with protein biogenesis factors and targeting of ribosomes synthesizing membrane proteins to the plasma membrane. The data provide insights into how the ribosome shapes composition and quality of the cellular proteome.

Protein domains can fold into stable tertiary structures while they are synthesized on the ribosome. We used a high-performance, reconstituted in vitro translation system to investigate the folding of a small five-helix protein domain—the N-terminal domain of Escherichia coli N5-glutamine methyltransferase HemK—in real time. Our observations show that cotranslational folding of the protein, which folds autonomously and rapidly in solution, proceeds through a compact, non-native conformation that forms within the peptide tunnel of the ribosome. The compact state rearranges into a native-like structure immediately after the full domain sequence has emerged from the ribosome. Both folding transitions are rate-limited by translation, allowing for quasi-equilibrium sampling of the conformational space restricted by the ribosome. Cotranslational folding may be typical of small, intrinsically rapidly folding protein domains.

Many proteins in the cell fold cotranslationally within the restricted space of the polypeptide exit tunnel or at the surface of the ribosome. A growing body of evidence suggests that the ribosome can alter the folding trajectory in many different ways. In this review, we summarize the recent examples of how translation affects folding of single-domain, multiple-domain and oligomeric proteins. The vectorial nature of translation, the spatial constraints of the exit tunnel, and the electrostatic properties of the ribosome-nascent peptide complex define the onset of early folding events. The ribosome can facilitate protein compaction, induce the formation of intermediates that are not observed in solution, or delay the onset of folding. Examples of single-domain proteins suggest that early compaction events can define the folding pathway for some types of domain structures. Folding of multi-domain proteins proceeds in a domain-wise fashion, with each domain having its role in stabilizing or destabilizing neighboring domains. Finally, the assembly of protein complexes can also begin cotranslationally. In all these cases, the ribosome helps the nascent protein to attain a native fold and avoid the kinetic traps of misfolding.

This review summarizes our current understanding of translation in prokaryotes, focusing on the mechanistic and structural aspects of each phase of translation: initiation, elongation, termination, and ribosome recycling. The assembly of the initiation complex provides multiple checkpoints for messenger RNA (mRNA) and start-site selection. Correct codon-anticodon interaction during the decoding phase of elongation results in major conformational changes of the small ribosomal subunit and shapes the reaction pathway of guanosine triphosphate (GTP) hydrolysis. The ribosome orchestrates proton transfer during peptide bond formation, but requires the help of elongation factor P (EF-P) when two or more consecutive Pro residues are to be incorporated. Understanding the choreography of transfer RNA (tRNA) and mRNA movements during translocation helps to place the available structures of translocation intermediates onto the time axis of the reaction pathway. The nascent protein begins to fold cotranslationally, in the constrained space of the polypeptide exit tunnel of the ribosome. When a stop codon is reached at the end of the coding sequence, the ribosome, assisted by termination factors, hydrolyzes the ester bond of the peptidyl-tRNA, thereby releasing the nascent protein. Following termination, the ribosome is dissociated into subunits and recycled into another round of initiation. At each step of translation, the ribosome undergoes dynamic fluctuations between different conformation states. The aim of this article is to show the link between ribosome structure, dynamics, and function.

Release factors RF1 and RF2 promote hydrolysis of peptidyl-tRNA during translation termination. The GTPase RF3 promotes recycling of RF1 and RF2. Using single molecule FRET and biochemical assays, we show that ribosome termination complexes that carry two factors, RF1–RF3 or RF2–RF3, are dynamic and fluctuate between non-rotated and rotated states, whereas each factor alone has its distinct signature on ribosome dynamics and conformation. Dissociation of RF1 depends on peptide release and the presence of RF3, whereas RF2 can dissociate spontaneously. RF3 binds in the GTP-bound state and can rapidly dissociate without GTP hydrolysis from termination complex carrying RF1. In the absence of RF1, RF3 is stalled on ribosomes if GTP hydrolysis is blocked. Our data suggest how the assembly of the ribosome–RF1–RF3–GTP complex, peptide release, and ribosome fluctuations promote termination of protein synthesis and recycling of the release factors., eLife digest Inside cells, molecular machines called ribosomes make proteins using messenger RNA as a template. However, the template contains more than just the information needed to create the protein. A ‘stop codon’ in the mRNA marks where the ribosome should stop. When this is reached a group of proteins called release factors removes the newly made protein from the ribosome. Bacteria typically have three types of release factors. RF1 and RF2 recognize the stop codon, and RF3 helps to release RF1 or RF2 from the ribosome so that it can be recycled to produce another protein. It was not fully understood how the release factors interact with the ribosome and how this terminates protein synthesis. Adio et al. used TIRF microscopy to study individual ribosomes from the commonly studied bacteria species Escherichia coli. This technique allows researchers to monitor movements of the ribosome and record how release factors bind to it. The results of the experiments performed by Adio et al. show that although RF1 and RF2 are very similar to each other, they interact with the ribosome in different ways. In addition, only RF1 relies upon RF3 to release it from the ribosome; RF2 can release itself. RF3 releases RF1 by forcing the ribosome to change shape. RF3 then uses energy produced by the breakdown of a molecule called GTP to help release itself from the ribosome. Most importantly, the findings presented by Adio et al. highlight that the movements of ribosomes and release factors during termination are only loosely coupled rather than occur in a set order. Other molecular machines are likely to work in a similar way. The results could also help us to understand the molecular basis of several human diseases, such as Duchenne muscular dystrophy and cystic fibrosis, that result from ribosomes not recognizing stop codons in the mRNA.

During translation termination in bacteria, the release factors RF1 and RF2 are recycled from the ribosome by RF3. While high-resolution structures of the individual termination factors on the ribosome exist, direct structural insight into how RF3 mediates dissociation of the decoding RFs has been lacking. Here we have used the Apidaecin 137 peptide to trap RF1 together with RF3 on the ribosome and visualize an ensemble of termination intermediates using cryo-electron microscopy. Binding of RF3 to the ribosome induces small subunit (SSU) rotation and swivelling of the head, yielding intermediate states with shifted P-site tRNAs and RF1 conformations. RF3 does not directly eject RF1 from the ribosome, but rather induces full rotation of the SSU that indirectly dislodges RF1 from its binding site. SSU rotation is coupled to the accommodation of the GTPase domain of RF3 on the large subunit (LSU), thereby promoting GTP hydrolysis and dissociation of RF3 from the ribosome., In bacteria, the process of translation termination is performed by three termination release factors RF1, RF2 and RF3. Here the authors provide detailed structural insights into the mechanism by which RF1 is dissociated from the ribosome by RF3 during termination.

Programmed -1 ribosomal frameshifting (-1PRF) is a recoding mechanism to make alternative proteins from a single mRNA transcript. -1PRF is stimulated by cis-acting signals in mRNA, a seven-nucleotide slippery sequence and a downstream secondary structure element, which is often a pseudoknot. In this study we engineered the frameshifting pseudoknot from the mouse mammary tumor virus to respond to a rationally designed small molecule naphthyridine carbamate tetramer (NCTn). We demonstrate that NCTn can stabilize the pseudoknot structure in mRNA and activate -1PRF both in vitro and in human cells. The results illustrate how NCTn-inducible -1PRF may serve as an important component of the synthetic biology toolbox for the precise control of gene expression using small synthetic molecules.

Highlights • –1PRF occurs when ribosomes move over a slippery sequence. • A frameshifting pseudoknot/stem-loop element stalls ribosomes in a metastable state. • –1PRF may contribute to the quality-control machinery in eukaryotes. • Trans-acting factors (proteins, miRNAs, or antibiotics) can modulate –1PRF., Programmed −1 ribosomal frameshifting (−1PRF) is an mRNA recoding event commonly utilized by viruses and bacteria to increase the information content of their genomes. Recent results have implicated −1PRF in quality control of mRNA and DNA stability in eukaryotes. Biophysical experiments demonstrated that the ribosome changes the reading frame while attempting to move over a slippery sequence of the mRNA – when a roadblock formed by a folded downstream segment in the mRNA stalls the ribosome in a metastable conformational state. The efficiency of −1PRF is modulated not only by cis-regulatory elements in the mRNA but also by trans-acting factors such as proteins, miRNAs, and antibiotics. These recent results suggest a molecular mechanism and new important cellular roles for −1PRF.

Cryo–electron microscopy shows how the ribosome prepares for take-off to bypass a noncoding mRNA stretch., Bypassing is a recoding event that leads to the translation of two distal open reading frames into a single polypeptide chain. We present the structure of a translating ribosome stalled at the bypassing take-off site of gene 60 of bacteriophage T4. The nascent peptide in the exit tunnel anchors the P-site peptidyl-tRNAGly to the ribosome and locks an inactive conformation of the peptidyl transferase center (PTC). The mRNA forms a short dynamic hairpin in the decoding site. The ribosomal subunits adopt a rolling conformation in which the rotation of the small subunit around its long axis causes the opening of the A-site region. Together, PTC conformation and mRNA structure safeguard against premature termination and read-through of the stop codon and reconfigure the ribosome to a state poised for take-off and sliding along the noncoding mRNA gap.

Summary Ribosome frameshifting during translation of bacterial dnaX can proceed via different routes, generating a variety of distinct polypeptides. Using kinetic experiments, we show that –1 frameshifting predominantly occurs during translocation of two tRNAs bound to the slippery sequence codons. This pathway depends on a stem-loop mRNA structure downstream of the slippery sequence and operates when aminoacyl-tRNAs are abundant. However, when aminoacyl-tRNAs are in short supply, the ribosome switches to an alternative frameshifting pathway that is independent of a stem-loop. Ribosome stalling at a vacant 0-frame A-site codon results in slippage of the P-site peptidyl-tRNA, allowing for –1-frame decoding. When the –1-frame aminoacyl-tRNA is lacking, the ribosomes switch into –2 frame. Quantitative mass spectrometry shows that the –2-frame product is synthesized in vivo . We suggest that switching between frameshifting routes may enrich gene expression at conditions of aminoacyl-tRNA limitation.

Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.

Elongation factor G (EF‐G) promotes the movement of two tRNAs and the mRNA through the ribosome in each cycle of peptide elongation. During translocation, the tRNAs transiently occupy intermediate positions on both small (30S) and large (50S) ribosomal subunits. How EF‐G and GTP hydrolysis control these movements is still unclear. We used fluorescence labels that specifically monitor movements on either 30S or 50S subunits in combination with EF‐G mutants and translocation‐specific antibiotics to investigate timing and energetics of translocation. We show that EF‐G–GTP facilitates synchronous movements of peptidyl‐tRNA on the two subunits into an early post‐translocation state, which resembles a chimeric state identified by structural studies. EF‐G binding without GTP hydrolysis promotes only partial tRNA movement on the 50S subunit. However, rapid 30S translocation and the concomitant completion of 50S translocation require GTP hydrolysis and a functional domain 4 of EF‐G. Our results reveal two distinct modes for utilizing the energy of EF‐G binding and GTP hydrolysis and suggest that coupling of GTP hydrolysis to translocation is mediated through rearrangements of the 30S subunit.

In all domains of life, selenocysteine (Sec) is delivered to the ribosome by selenocysteine-specific tRNA (tRNASec) with the help of a specialized translation factor, SelB in bacteria. Sec-tRNASec recodes a UGA stop codon next to a downstream mRNA stem-loop. Here we present the structures of six intermediates on the pathway of UGA recoding in Escherichia coli by single-particle cryo-electron microscopy. The structures explain the specificity of Sec-tRNASec binding by SelB and show large-scale rearrangements of Sec-tRNASec. Upon initial binding of SelB-Sec-tRNASec to the ribosome and codon reading, the 30S subunit adopts an open conformation with Sec-tRNASec covering the sarcin-ricin loop (SRL) on the 50S subunit. Subsequent codon recognition results in a local closure of the decoding site, which moves Sec-tRNASec away from the SRL and triggers a global closure of the 30S subunit shoulder domain. As a consequence, SelB docks on the SRL, activating the GTPase of SelB. These results reveal how codon recognition triggers GTPase activation in translational GTPases.

In each round of translation elongation, tRNAs and mRNA move within the ribosome by one codon at a time. tRNA-mRNA translocation is promoted by elongation factor G (EF-G) at the cost of GTP hydrolysis. The key questions for understanding translocation are how and when the tRNAs move and how EF-G coordinates motions of the ribosomal subunits with tRNA movement. Here we present 2 recent papers which describe the choreography of movements over the whole trajectory of translocation. We present the view that EF-G accelerates translocation by promoting the steps that lead to GTPase-dependent ribosome unlocking. EF-G facilitates the formation of the rotated state of the ribosome and uncouples the backward motions of the ribosomal subunits, forming an open conformation in which the tRNAs can rapidly move. Ribosome dynamics are important not only in translocation, but also in recoding events, such as frameshifting and bypassing, and mediate sensitivity to antibiotics.

During protein synthesis, tRNAs move from the ribosome's aminoacyl to peptidyl to exit sites. Here we investigate conformational motions during spontaneous translocation, using molecular dynamics simulations of 13 intermediate-translocation-state models obtained by combining Escherichia coli ribosome crystal structures with cryo-EM data. Resolving fast transitions between states, we find that tRNA motions govern the transition rates within the pre- and post-translocation states. Intersubunit rotations and L1-stalk motion exhibit fast intrinsic submicrosecond dynamics. The L1 stalk drives the tRNA from the peptidyl site and links intersubunit rotation to translocation. Displacement of tRNAs is controlled by 'sliding' and 'stepping' mechanisms involving conserved L16, L5 and L1 residues, thus ensuring binding to the ribosome despite large-scale tRNA movement. Our results complement structural data with a time axis, intrinsic transition rates and molecular forces, revealing correlated functional motions inaccessible by other means.

The elongation phase of translation is promoted by three universal elongation factors, EF-Tu, EF-Ts, and EF-G in bacteria and their homologs in archaea and eukaryotes. Recent findings demonstrate that the translation of a subset of mRNAs requires a fourth elongation factor, EF-P in bacteria or the homologs factors a/eIF5A in other kingdoms of life. EF-P prevents the ribosome from stalling during the synthesis of proteins containing consecutive Pro residues, such as PPG, PPP, or longer Pro clusters. The efficient and coordinated synthesis of such proteins is required for bacterial growth, motility, virulence, and stress response. EF-P carries a unique post-translational modification, which contributes to its catalytic proficiency. The modification enzymes, which are lacking in higher eukaryotes, provide attractive new targets for the development of new, highly specific antimicrobials.

In all contemporary organisms, the active site of the ribosome--the peptidyl transferase center--catalyzes two distinct reactions, peptide bond formation between peptidyl-tRNA and aminoacyl-tRNA as well as the hydrolysis of peptidyl-tRNA with the help of a release factor. However, when provided with appropriate substrates, ribosomes can also catalyze a broad range of other chemical reaction, which provides the basis for orthogonal translation and synthesis of alloproteins from unnatural building blocks. Advances in understanding the mechanisms of the two ubiquitous reactions, the peptide bond formation and peptide release, provide insights into the versatility of the active site of the ribosome. Release factors 1 and 2 and elongation factor P are auxiliary factors that augment the intrinsic catalytic activity of the ribosome in special cases.

Translating Polyproline Translation of messenger RNA into protein is carried out by the ribosome, together with a variety of accessory factors, which offer the potential for regulation of this critical step in gene expression (see the Perspective by Buskirk and Green ). Ude et al. (p. 82 , published online 13 December), using bacterial genetics and an in vitro reconstituted translation system, and Doerfel et al. (p. 85 , published online 13 December), using a model assay for peptide bond formation, find that the universally conserved bacterial elongation factor P (EF-P) (which is orthologous to the archaeal and eukaryotic initiation factor 5A) is required for the efficient translation of polyproline-containing polypeptides. Such short polyproline stretches (with runs of two, three, or more proline residues) would otherwise cause ribosomal stalling.

The cytotoxin colicin E3 targets the 30S subunit of bacterial ribosomes and specifically cleaves 16S rRNA at the decoding centre, thereby inhibiting translation. Although the cleavage site is well known, it is not clear which step of translation is inhibited. We studied the effects of colicin E3 cleavage on ribosome functions by analysing individual steps of protein synthesis. We find that the cleavage affects predominantly the elongation step. The inhibitory effect of colicin E3 cleavage originates from the accumulation of sequential impaired decoding events, each of which results in low occupancy of the A site and, consequently, decreasing yield of elongating peptide. The accumulation leads to an almost complete halt of translation after reading of a few codons. The cleavage of 16S rRNA does not impair monitoring of codon–anticodon complexes or GTPase activation during elongation-factor Tu-dependent binding of aminoacyl-tRNA, but decreases the stability of the codon–recognition complex and slows down aminoacyl-tRNA accommodation in the A site. The tRNA–mRNA translocation is faster on colicin E3-cleaved than on intact ribosomes and is less sensitive to inhibition by the antibiotic viomycin.

The ribosome stalls on translation of polyproline sequences due to inefficient peptide bond formation between consecutive prolines. The translation factor EF-P is able to alleviate this stalling by accelerating Pro-Pro formation. However, the mechanism by which EF-P recognizes the stalled complexes and accelerates peptide bond formation is not known. Here, we use genetic code reprogramming through a flexible in-vitro translation (FIT) system to investigate how mutations in tRNAPro affect EF-P function. We show that the 9-nt D-loop closed by the stable D-stem sequence in tRNAPro is a crucial recognition determinant for EF-P. Such D-arm structures are shared only among the tRNAPro isoacceptors and tRNAfMet in Escherichia coli, and the D-arm of tRNAfMet is essential for EF-P-induced acceleration of fMet–puromycin formation. Thus, the activity of EF-P is controlled by recognition elements in the tRNA D-arm., Ribosomes tend to stall during the translation of consecutive proline residues, which can be rescued by the co-translational factor EF-P. Here the authors identify a structural element of tRNAPro responsible for specific recognition by EF-P and stimulation of Pro-Pro peptide bond formation.

The ribosome is the molecular machine responsible for protein synthesis in all living organisms. Its catalytic core, the peptidyl transferase center (PTC), is built of rRNA, although several proteins reach close to the inner rRNA shell. In the Escherichia coli ribosome, the flexible N-terminal tail of the ribosomal protein L27 contacts the A- and P-site tRNA. Based on computer simulations of the PTC and on previous biochemical evidence, the N-terminal α-amino group of L27 was suggested to take part in the peptidyl-transfer reaction. However, the contribution of this group to catalysis has not been tested experimentally. Here we investigate the role of L27 in peptide-bond formation using fast kinetics approaches. We show that the rate of peptide-bond formation at physiological pH, both with aminoacyl-tRNA or with the substrate analog puromycin, is independent of the presence of L27; furthermore, translation of natural mRNAs is only marginally affected in the absence of L27. The pH dependence of the puromycin reaction is unaltered in the absence of L27, indicating that the N-terminal α-amine is not the ionizing group taking part in catalysis. Likewise, L27 is not required for the peptidyl-tRNA hydrolysis during termination. Thus, apart from the known effect on subunit association, which most likely explains the phenotype of the deletion strains, L27 does not appear to be a key player in the core mechanism of peptide-bond formation on the ribosome.

Speed and accuracy of protein synthesis are fundamental parameters for the fitness of living cells, the quality control of translation, and the evolution of ribosomes. The ribosome developed complex mechanisms that allow for a uniform recognition and selection of any cognate aminoacyl-tRNA (aa-tRNA) and discrimination against any near-cognate aa-tRNA, regardless of the nature or position of the mismatch. This review describes the principles of the selection—kinetic partitioning and induced fit—and discusses the relationship between speed and accuracy of decoding, with a focus on bacterial translation. The translational machinery apparently has evolved towards high speed of translation at the cost of fidelity.

During protein synthesis, the nascent peptide chain traverses the peptide exit tunnel of the ribosome. We monitor the co-translational movement of the nascent peptide using a fluorescent probe attached to the N-terminus of the nascent chain. Due to fluorophore quenching, the time-dependent fluorescence signal emitted by an individual peptide is determined by co-translational events, such as secondary structure formation and peptide-tunnel interactions. To obtain information on these individual events, the measured ensemble fluorescence signal has to be decomposed into position-dependent intensities. Here, we describe mRNA translation as a Markov process with specific fluorescence intensities assigned to the different states of the process. Combining the computed stochastic time evolution of the translation process with a sequence of observed ensemble fluorescence time courses, we compute the unknown position-specific intensities and obtain detailed information on the kinetics of the translation process. In particular, we find that translation of poly(U) mRNAs dramatically slows down at the fourth UUU codon. The method presented here detects subtle differences in the position-specific fluorescence intensities and thus provides a novel approach to study translation kinetics in ensemble experiments.

During protein synthesis, aminoacyl-tRNA (aa-tRNA) and release factors 1 and 2 (RF1 and RF2) have to bind at the catalytic center of the ribosome on the 50S subunit where they take part in peptide bond formation or peptidyl-tRNA hydrolysis, respectively. Computer simulations of aa-tRNA movement into the catalytic site (accommodation) suggested that three nucleotides of 23S rRNA, U2492, C2556, and C2573, form a “gate” at which aa-tRNA movement into the A site is retarded. Here we examined the role of nucleotides C2573 of 23S rRNA, a part of the putative accommodation gate, and of the neighboring A2572 for aa-tRNA binding followed by peptide bond formation and for the RF2-dependent peptide release. Mutations at the two positions did not affect aa-tRNA accommodation, peptide bond formation, or the fidelity of aa-tRNA selection, but impaired RF2-catalyzed peptide release. The data suggest that the ribosome is a robust machine that allows rapid aa-tRNA accommodation despite the defects at the accommodation gate. In comparison, peptide release by RF2 appears more sensitive to these mutations, due to slower accommodation of the factor or effects on RF2 positioning in the A site.

The translation initiation efficiency of a given mRNA is determined by its translation initiation region (TIR). mRNAs are selected into 30S initiation complexes according to the strengths of the secondary structure of the TIR, the pairing of the Shine-Dalgarno sequence with 16S rRNA, and the interaction between initiator tRNA and the start codon. Here, we show that the conversion of the 30S initiation complex into the translating 70S ribosome constitutes another important mRNA control checkpoint. Kinetic analysis reveals that 50S subunit joining and dissociation of IF3 are strongly influenced by the nature of the codon used for initiation and the structural elements of the TIR. Coupling between the TIR and the rate of 70S initiation complex formation involves IF3- and IF1-induced rearrangements of the 30S subunit, providing a mechanism by which the ribosome senses the TIR and determines the efficiency of translational initiation of a particular mRNA.

Peptide bond formation on the ribosome takes place in an active site composed of RNA. Recent progress of structural, biochemical, and computational approaches has provided a fairly detailed picture of the catalytic mechanism of the reaction. The ribosome accelerates peptide bond formation by lowering the activation entropy of the reaction due to positioning the two substrates, ordering water in the active site, and providing an electrostatic network that stabilizes the reaction intermediates. Proton transfer during the reaction appears to be promoted by a concerted proton shuttle mechanism that involves ribose hydroxyl groups on the tRNA substrate.

During the translocation step of protein synthesis, a complex of two transfer RNAs bound to messenger RNA (tRNA-mRNA) moves through the ribosome. The reaction is promoted by an elongation factor, called EF-G in bacteria, which, powered by GTP hydrolysis, induces an open, unlocked conformation of the ribosome that allows for spontaneous tRNA-mRNA movement. Here we show that, in the absence of EF-G, there is spontaneous backward movement, or retrotranslocation, of two tRNAs bound to mRNA. Retrotranslocation is driven by the gain in affinity when a cognate E-site tRNA moves into the P site, which compensates the affinity loss accompanying the movement of peptidyl-tRNA from the P to the A site. These results lend support to the diffusion model of tRNA movement during translocation. In the cell, tRNA movement is biased in the forward direction by EF-G, which acts as a Brownian ratchet and prevents backward movement.

The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs. Recent structural work has visualized intermediates of translocation induced by elongation factor G (EF-G) with tRNAs trapped in chimeric states with respect to 30S and 50S ribosomal subunits. The functional role of the chimeric states is not known. Here we follow the formation of translocation intermediates by single-molecule fluorescence resonance energy transfer. Using EF-G mutants, a non-hydrolysable GTP analogue, and fusidic acid, we interfere with either translocation or EF-G release from the ribosome and identify several rapidly interconverting chimeric tRNA states on the reaction pathway. EF-G engagement prevents backward transitions early in translocation and increases the fraction of ribosomes that rapidly fluctuate between hybrid, chimeric and posttranslocation states. Thus, the engagement of EF-G alters the energetics of translocation towards a flat energy landscape, thereby promoting forward tRNA movement., EF-G enhances the rate of tRNA–mRNA translocation on the ribosome. Here the authors use single-molecule FRET to follow tRNA translocation in real time, identifying new chimeric intermediates and suggesting how EF-G binding and GTP hydrolysis change the energetic landscape of translocation to accelerate forward tRNA movement.

The ribosome is a molecular machine that synthesizes polypeptides from aminoacyl-tRNAs according to the sequence of the mRNA template. Codon reading by the anticodon of tRNA is controlled by a network of ribosome contacts that are specific for each position of the codon-anticodon duplex and involve A-minor RNA interactions. Rapid and accurate tRNA selection is accomplished by switching the conformation of the decoding site between accepting and rejecting mode, regardless of the thermodynamic stability of the respective codon-anticodon complexes or their interactions at the decoding site. The forward reactions are particularly sensitive to mismatches and determine the variations in the extent of misreading of near-cognate codons, both during initial selection and proofreading. This review emphasizes the progress made in understanding the mechanisms that determine recognition and selection of tRNA by the translational machinery.