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Ribosomal action is facilitated by the orchestrated work of trans-acting factors and ribosomal elements, which are subject to regulatory events, often involving phosphorylation. One such element is the ribosomal P-stalk, which plays a dual function: it activates translational GTPases, which support basic ribosomal functions, and interacts with the Gcn2 kinase, linking the ribosomes to the ISR pathway. We show that P-stalk proteins, which form a pentamer, exist in the cell exclusively in a phosphorylated state at five C-terminal domains (CTDs), ensuring optimal translation (speed and accuracy) and may play a role in the timely regulation of the Gcn2-dependent stress response. Phosphorylation of the CTD induces a structural transition from a collapsed to a coil-like structure, and the CTD gains conformational freedom, allowing specific but transient binding to various protein partners, optimizing the ribosome action. The report reveals a unique feature of the P-stalk proteins, indicating that, unlike most ribosomal proteins, which are regulated by phosphorylation in an on/off manner, the P-stalk proteins exist in a constantly phosphorylated state, which optimizes their interaction with auxiliary factors., (© 2024. The Author(s).)

EF-P is a translation factor that facilitates the formation of peptide bonds between consecutive prolines. Using FRET between EF-P and ribosomal protein bL33, we studied dynamics and specificity of EF-P binding to the ribosome. Our findings reveal that EF-P rapidly scans for a free E site and can bind to any ribosome containing a P-site tRNA, regardless of the ribosome's functional state. The interaction with uL1 is essential for EF-P binding, while the β-Lys modification of EF-P doubles the association rate. Specific interactions with the D-loop of tRNAPro or tRNAfMet and via the β-Lys group with the tRNA in the peptidyl transferase center reduce the rate of EF-P dissociation from the ribosome, providing the specificity for complexes that need help in catalyzing peptide bond formation. The nature of the E-site codon has little effect on EF-P binding kinetics. Although EF-P dissociation is reduced upon recognizing its correct tRNA substrate, it remains sufficiently rapid compared to tRNA translocation and does not affect the translocation rate. These results highlight the importance of EF-P's scanning-engagement mechanism for dynamic substrate recognition during rapid translation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The selection of an open reading frame (ORF) for translation of eukaryotic mRNA relies on remodeling of the scanning 48S initiation complex into an elongation-ready 80S ribosome. Using cryo-electron microscopy, we visualize the key commitment steps orchestrating 48S remodeling in humans. The mRNA Kozak sequence facilitates mRNA scanning in the 48S open state and stabilizes the 48S closed state by organizing the contacts of eukaryotic initiation factors (eIFs) and ribosomal proteins and by reconfiguring mRNA structure. GTPase-triggered large-scale fluctuations of 48S-bound eIF2 facilitate eIF5B recruitment, transfer of initiator tRNA from eIF2 to eIF5B and the release of eIF5 and eIF2. The 48S-bound multisubunit eIF3 complex controls ribosomal subunit joining by coupling eIF exchange to gradual displacement of the eIF3c N-terminal domain from the intersubunit interface. These findings reveal the structural mechanism of ORF selection in human cells and explain how eIF3 could function in the context of the 80S ribosome., (© 2024. The Author(s).)

In this Review, we explore natural product antibiotics that do more than simply inhibit an active site of an essential enzyme. We review these compounds to provide inspiration for the design of much-needed new antibacterial agents, and examine the complex mechanisms that have evolved to effectively target bacteria, including covalent binders, inhibitors of resistance, compounds that utilize self-promoted entry, those that evade resistance, prodrugs, target corrupters, inhibitors of 'undruggable' targets, compounds that form supramolecular complexes, and selective membrane-acting agents. These are exemplified by β-lactams that bind covalently to inhibit transpeptidases and β-lactamases, siderophore chimeras that hijack import mechanisms to smuggle antibiotics into the cell, compounds that are activated by bacterial enzymes to produce reactive molecules, and antibiotics such as aminoglycosides that corrupt, rather than merely inhibit, their targets. Some of these mechanisms are highly sophisticated, such as the preformed β-strands of darobactins that target the undruggable β-barrel chaperone BamA, or teixobactin, which binds to a precursor of peptidoglycan and then forms a supramolecular structure that damages the membrane, impeding the emergence of resistance. Many of the compounds exhibit more than one notable feature, such as resistance evasion and target corruption. Understanding the surprising complexity of the best antimicrobial compounds provides a roadmap for developing novel compounds to address the antimicrobial resistance crisis by mining for new natural products and inspiring us to design similarly sophisticated antibiotics., (© 2024. Springer Nature Limited.)

The mRNA coding sequence defines not only the amino acid sequence of the protein, but also the speed at which the ribosomes move along the mRNA while making the protein. The non-uniform local kinetics - denoted as translational rhythm - is similar among mRNAs coding for related protein folds. Deviations from this conserved rhythm can result in protein misfolding. In this review we summarize the experimental evidence demonstrating how local translation rates affect cotranslational protein folding, with the focus on the synonymous codons and patches of charged residues in the nascent peptide as best-studied examples. Alterations in nascent protein conformations due to disturbed translational rhythm can persist off the ribosome, as demonstrated by the effects of synonymous codon variants of several disease-related proteins. Charged amino acid patches in nascent chains also modulate translation and cotranslational protein folding, and can abrogate translation when placed at the N-terminus of the nascent peptide. During cotranslational folding, incomplete nascent chains navigate through a unique conformational landscape in which earlier intermediate states become inaccessible as the nascent peptide grows. Precisely tuned local translation rates, as well as interactions with the ribosome, guide the folding pathway towards the native structure, whereas deviations from the natural translation rhythm may favor pathways leading to trapped misfolded states. Deciphering the 'folding code' of the mRNA will contribute to understanding the diseases caused by protein misfolding and to rational protein design., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The ribosome can slide along mRNA without establishing codon-anticodon interactions. This movement can be regulated (programmed) by the elements encoded in the mRNA, as observed in bypassing of non-coding gap in gene 60 of bacteriophage T4, or occur spontaneously, such as during traversal by the 70S ribosome of the 3'UTRs or upon re-initiation on bacterial polycistronic genes. In this study, we investigate the kinetic mechanism underlying the programmed and spontaneous ribosome sliding. We show that the translation rate of gene 60 mRNA decreases as the ribosome approaches the take-off site, especially when the KKYK regulatory sequence in the nascent peptide reaches the constriction site in the ribosome exit tunnel. However, efficiency of bypassing increases when the ribosome traverses the gap quickly. With the non-coding gap exceeding the natural 50 nt, the processivity of sliding remains high up to 56 nt, but drops sharply beyond that due to the loss of mRNA elements support. Sliding efficiency is temperature-dependent; while temperature regulates the number of ribosomes initiating programmed bypassing, traversing the long gaps becomes increasingly unfavorable at lower temperatures. This data offers novel insights into the kinetic determinants of programmed and spontaneous ribosome sliding along the mRNA., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The innate immune system, particularly the interferon (IFN) system, constitutes the initial line of defense against viral infections. IFN signaling induces the expression of interferon-stimulated genes (ISGs), and their products frequently restrict viral infection. Retroviruses like the human immunodeficiency viruses and the human T-lymphotropic viruses cause severe human diseases and are targeted by ISG-encoded proteins. Here, we discuss ISGs that inhibit the translation of retroviral mRNAs and thereby retrovirus propagation. The Schlafen proteins degrade cellular tRNAs and rRNAs needed for translation. Zinc Finger Antiviral Protein and RNA-activated protein kinase inhibit translation initiation factors, and Shiftless suppresses translation recoding essential for the expression of retroviral enzymes. We outline common mechanisms that underlie the antiviral activity of multifunctional ISGs and discuss potential antiretroviral therapeutic approaches based on the mode of action of these ISGs.

The interferon-induced host cell protein Shiftless (SFL) inhibits -1 programmed ribosomal frameshifting (-1PRF) required for the expression of HIV-1 Gal-Pol and the formation of infectious HIV-1 particles. However, the specific regions in SFL required for antiviral activity and the mechanism by which SFL inhibits -1PRF remain unclear. Employing alanine scanning mutagenesis, we found that basic amino acids in the predicted zinc ribbon motif of SFL are essential for the suppression of Gag-Pol expression but dispensable for anti-HIV-1 activity. We have shown that SFL inhibits the expression of the murine leukemia virus (MLV) Gag-Pol polyprotein and the formation of infectious MLV particles, although Gag-Pol expression of MLV is independent of -1PRF but requires readthrough of a stop codon. These findings indicate that SFL might inhibit HIV-1 infection by more than one mechanism and that SFL might target programmed translational readthrough as well as -1PRF signals, both of which are regulated by mRNA secondary structure elements.

Processive enzymes like polymerases or ribosomes are often studied in bulk experiments by monitoring time-dependent signals, such as fluorescence time traces. However, due to biomolecular process stochasticity, ensemble signals may lack the distinct features of single-molecule signals. Here, we demonstrate that, under certain conditions, bulk signals from processive reactions can be decomposed to unveil hidden information about individual reaction steps. Using mRNA translation as a case study, we show that decomposing a noisy ensemble signal generated by the translation of mRNAs with more than a few codons is an ill-posed problem, addressable through Tikhonov regularization. We apply our method to the fluorescence signatures of in-vitro translated LepB mRNA and determine codon-position dependent translation rates and corresponding state-specific fluorescence intensities. We find a significant change in fluorescence intensity after the fourth and the fifth peptide bond formation, and show that both codon position and encoded amino acid have an effect on the elongation rate. This demonstrates that our approach enhances the information content extracted from bulk experiments, thereby expanding the range of these time- and cost-efficient methods., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Haase et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Svetlov et al. identify the enzyme peptidyl-tRNA hydrolase as a ribosome-associated quality-control factor that promotes hydrolysis of the dislodged peptidyl-tRNA, which helps to recycle ribosomal subunits blocked by truncated nascent chains in bacteria., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

During protein synthesis, the growing nascent peptide chain moves inside the polypeptide exit tunnel of the ribosome from the peptidyl transferase center towards the exit port where it emerges into the cytoplasm. The ribosome defines the unique energy landscape of the pioneering round of protein folding. The spatial confinement and the interactions of the nascent peptide with the tunnel walls facilitate formation of secondary structures, such as α-helices. The vectorial nature of protein folding inside the tunnel favors local intra- and inter-molecular interactions, thereby inducing cotranslational folding intermediates that do not form upon protein refolding in solution. Tertiary structures start to fold in the lower part of the tunnel, where interactions with the ribosome destabilize native protein folds. The present review summarizes the recent progress in understanding the driving forces of nascent protein folding inside the tunnel and at the surface of the ribosome., Competing Interests: Declaration of competing interest None., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

N 6 -methyladenosine (m 6 A) is an abundant, dynamic mRNA modification that regulates key steps of cellular mRNA metabolism. m 6 A in the mRNA coding regions inhibits translation elongation. Here, we show how m 6 A modulates decoding in the bacterial translation system using a combination of rapid kinetics, smFRET and single-particle cryo-EM. We show that, while the modification does not impair the initial binding of aminoacyl-tRNA to the ribosome, in the presence of m 6 A fewer ribosomes complete the decoding process due to the lower stability of the complexes and enhanced tRNA drop-off. The mRNA codon adopts a π-stacked codon conformation that is remodeled upon aminoacyl-tRNA binding. m 6 A does not exclude canonical codon-anticodon geometry, but favors alternative more dynamic conformations that are rejected by the ribosome. These results highlight how modifications outside the Watson-Crick edge can still interfere with codon-anticodon base pairing and complex recognition by the ribosome, thereby modulating the translational efficiency of modified mRNAs., (© 2023. Springer Nature Limited.)

Faithful translation of messenger RNA (mRNA) into protein is essential to maintain protein homeostasis in the cell. Spontaneous translation errors are very rare due to stringent selection of cognate aminoacyl transfer RNAs (tRNAs) and the tight control of the mRNA reading frame by the ribosome. Recoding events, such as stop codon readthrough, frameshifting, and translational bypassing, reprogram the ribosome to make intentional mistakes and produce alternative proteins from the same mRNA. The hallmark of recoding is the change of ribosome dynamics. The signals for recoding are built into the mRNA, but their reading depends on the genetic makeup of the cell, resulting in cell-specific changes in expression programs. In this review, I discuss the mechanisms of canonical decoding and tRNA-mRNA translocation; describe alternative pathways leading to recoding; and identify the links among mRNA signals, ribosome dynamics, and recoding.

In each round of translation elongation, the ribosome translocates along the mRNA by precisely one codon. Translocation is promoted by elongation factor G (EF-G) in bacteria (eEF2 in eukaryotes) and entails a number of precisely-timed large-scale structural rearrangements. As a rule, the movements of the ribosome, tRNAs, mRNA and EF-G are orchestrated to maintain the exact codon-wise step size. However, signals in the mRNA, as well as environmental cues, can change the timing and dynamics of the key rearrangements leading to recoding of the mRNA into production of trans-frame peptides from the same mRNA. In this review, we discuss recent advances on the mechanics of translocation and reading frame maintenance. Furthermore, we describe the mechanisms and biological relevance of non-canonical translocation pathways, such as hungry and programmed frameshifting and translational bypassing, and their link to disease and infection., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Protein synthesis is crucial for cell growth and survival yet one of the most energy-consuming cellular processes. How, then, do cells sustain protein synthesis under starvation conditions when energy is limited? To accelerate the translocation of mRNA-tRNAs through the ribosome, bacterial elongation factor G (EF-G) hydrolyzes energy-rich guanosine triphosphate (GTP) for every amino acid incorporated into a protein. Here, we identify an EF-G paralog-EF-G2-that supports translocation without hydrolyzing GTP in the gut commensal bacterium Bacteroides thetaiotaomicron. EF-G2's singular ability to sustain protein synthesis, albeit at slow rates, is crucial for bacterial gut colonization. EF-G2 is ~10-fold more abundant than canonical EF-G1 in bacteria harvested from murine ceca and, unlike EF-G1, specifically accumulates during carbon starvation. Moreover, we uncover a 26-residue region unique to EF-G2 that is essential for protein synthesis, EF-G2 dissociation from the ribosome, and responsible for the absence of GTPase activity. Our findings reveal how cells curb energy consumption while maintaining protein synthesis to advance fitness in nutrient-fluctuating environments., (© 2022 The Authors.)

Processing of newly synthesized polypeptides is essential for protein homeostasis and cell viability. In bacteria and eukaryotic organelles, all proteins are synthesized with formylmethionine at their N-terminus. As the nascent peptide emerges from the ribosome during translation, the formyl group is removed by peptide deformylase (PDF), an enzyme that belongs to the family of ribosome-associated protein biogenesis factors (RPBs). Because PDF is essential in bacteria but not in humans (except for the PDF homolog acting in mitochondria), the bacterial enzyme is a promising antimicrobial drug target. While much of the mechanistic work on PDF was carried out using model peptides in solution, understanding the mechanism of PDF in cells and developing effective PDF inhibitors requires experiments with its native cellular substrates, i.e., ribosome-nascent chain complexes. Here, we describe protocols to purify PDF from Escherichia coli and to test its deformylation activity on the ribosome in multiple-turnover and single-round kinetic regimes as well as in binding assays. These protocols can be used to test PDF inhibitors, to study the peptide specificity of PDF and its interplay with other RPBs, as well as to compare the activity and specificity of bacterial and mitochondrial PDFs., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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-tRNA Cys into a model protein using a reconstituted in-vitro translation system. We show that the modified Cys-tRNA Cys 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-tRNA Cys 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., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

The interferon-induced host cell protein shiftless (SFL) was reported to inhibit human immunodeficiency virus (HIV) infection by blocking the -1 programmed ribosomal frameshifting (-1PRF) required for expression of the Gag-Pol polyprotein. However, it is not clear how SFL inhibits -1PRF. To address this question, we focused on a 36 amino acids comprising region (termed required for antiviral activity (RAA)) that is essential for suppression of -1PRF and HIV infection and is missing from SFL short (SFLS), a splice variant of SFL with unknown function. Here, we confirm that SFL, but not SFLS, inhibits HIV -1PRF and show that inhibition is cell-type-independent. Mutagenic and biochemical analyses demonstrated that the RAA region is required for SFL self-interactions and confirmed that it is necessary for ribosome association and binding to the HIV RNA. Analysis of SFL mutants with six consecutive amino-acids-comprising deletions in the RAA region suggests effects on binding to the HIV RNA, complete inhibition of -1PRF, inhibition of Gag-Pol expression, and antiviral activity. In contrast, these amino acids did not affect SFL expression and were partially dispensable for SFL self-interactions and binding to the ribosome. Collectively, our results support the notion that SFL binds to the ribosome and the HIV RNA in order to block -1PRF and HIV infection, and suggest that the multimerization of SFL may be functionally important.

Translational 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 has been identified in several eukaryotic organisms; however, its biological significance and mechanism remain unclear. Here, we quantify TR of several candidate genes in Drosophila melanogaster and characterize the regulation of TR in the large Maf transcription factor Traffic jam (Tj). Using CRISPR/Cas9-generated mutant flies, we show that the TR-generated Tj isoform is expressed in a subset of neural cells of the central nervous system and is excluded from the somatic cells of gonads. Control of TR in Tj is critical for preservation of neuronal integrity and maintenance of reproductive health. The tissue-specific distribution of a release factor splice variant, eRF1H, plays a critical role in modulating differential TR of leaky stop codon contexts. Fine-tuning of gene regulatory functions of transcription factors by TR provides a potential mechanism for cell-specific regulation of gene expression., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2-GTP-Met-tRNAiMet and eIF3. The 'open' 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The 'closed' form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nascent polypeptides emerging from the ribosome during translation are rapidly scanned and processed by ribosome-associated protein biogenesis factors (RPBs). RPBs cleave the N-terminal formyl and methionine groups, assist cotranslational protein folding, and sort the proteins according to their cellular destination. Ribosomes translating inner-membrane proteins are recognized and targeted to the translocon with the help of the signal recognition particle, SRP, and SRP receptor, FtsY. The growing nascent peptide is then inserted into the phospholipid bilayer at the translocon, an inner-membrane protein complex consisting of SecY, SecE, and SecG. Folding of membrane proteins requires that transmembrane helices (TMs) attain their correct topology, the soluble domains are inserted at the correct (cytoplasmic or periplasmic) side of the membrane, and - for polytopic membrane proteins - the TMs find their interaction partner TMs in the phospholipid bilayer. This review describes the recent progress in understanding how growing nascent peptides are processed and how inner-membrane proteins are targeted to the translocon and find their correct orientation at the membrane, with the focus on biophysical approaches revealing the dynamics of the process. We describe how spontaneous fluctuations of the translocon allow diffusion of TMs into the phospholipid bilayer and argue that the ribosome orchestrates cotranslational targeting not only by providing the binding platform for the RPBs or the translocon, but also by helping the nascent chains to find their correct orientation in the membrane. Finally, we present the auxiliary role of YidC as a chaperone for inner-membrane proteins. We show how biophysical approaches provide new insights into the dynamics of membrane protein biogenesis and raise new questions as to how translation modulates protein folding., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mercier, Wang, Bögeholz, Wintermeyer and Rodnina.)

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., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Synthesis of bacterial proteins on the ribosome starts with a formylated methionine. Removal of the N-terminal formyl group is essential and is carried out by peptide deformylase (PDF). Deformylation occurs co-translationally, shortly after the nascent-chain emerges from the ribosomal exit tunnel, and is necessary to allow for further N-terminal processing. Here we describe the kinetic mechanism of deformylation by PDF of ribosome-bound nascent-chains and show that PDF binding to and dissociation from ribosomes is rapid, allowing for efficient scanning of formylated substrates in the cell. The rate-limiting step in the PDF mechanism is a conformational rearrangement of the nascent-chain that takes place after cleavage of the formyl group. Under conditions of ongoing translation, the nascent-chain is deformylated rapidly as soon as it becomes accessible to PDF. Following deformylation, the enzyme is slow in releasing the deformylated nascent-chain, thereby delaying further processing and potentially acting as an early chaperone that protects short nascent chains before they reach a length sufficient to recruit other protein biogenesis factors., (© 2021. The Author(s).)

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., (© 2021. The Author(s).)

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., (© 2021 Belardinelli et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Brr2 is an essential Ski2-like RNA helicase that exhibits a unique structure among the spliceosomal helicases. Brr2 harbors a catalytically active N-terminal helicase cassette and a structurally similar but enzymatically inactive C-terminal helicase cassette connected by a linker region. Both cassettes contain a nucleotide-binding pocket, but it is unclear whether nucleotide binding in these two pockets is related. Here we use biophysical and computational methods to delineate the functional connectivity between the cassettes and determine whether occupancy of one nucleotide-binding site may influence nucleotide binding at the other cassette. Our results show that Brr2 exhibits high specificity for adenine nucleotides, with both cassettes binding ADP tighter than ATP. Adenine nucleotide affinity for the inactive C-terminal cassette is more than two orders of magnitude higher than that of the active N-terminal cassette, as determined by slow nucleotide release. Mutations at the intercassette surfaces and in the connecting linker diminish the affinity of adenine nucleotides for both cassettes. Moreover, we found that abrogation of nucleotide binding at the C-terminal cassette reduces nucleotide binding at the N-terminal cassette 70 Å away. Molecular dynamics simulations identified structural communication lines that likely mediate these long-range allosteric effects, predominantly across the intercassette interface. Together, our results reveal intricate networks of intramolecular interactions in the complex Brr2 RNA helicase, which fine-tune its nucleotide affinities and which could be exploited to regulate enzymatic activity during splicing., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Aminoglycoside antibiotics target the ribosome and induce mistranslation, yet which translation errors induce bacterial cell death is unclear. The analysis of cellular proteins by quantitative mass spectrometry shows that bactericidal aminoglycosides induce not only single translation errors, but also clusters of errors in full-length proteins in vivo with as many as four amino acid substitutions in a row. The downstream errors in a cluster are up to 10,000-fold more frequent than the first error and independent of the intracellular aminoglycoside concentration. The prevalence, length, and composition of error clusters depends not only on the misreading propensity of a given aminoglycoside, but also on its ability to inhibit ribosome translocation along the mRNA. Error clusters constitute a distinct class of misreading events in vivo that may provide the predominant source of proteotoxic stress at low aminoglycoside concentration, which is particularly important for the autocatalytic uptake of the drugs.

The guided entry of tail-anchored proteins (GET) pathway assists in the posttranslational delivery of tail-anchored proteins, containing a single C-terminal transmembrane domain, to the ER. Here we uncover how the yeast GET pathway component Get4/5 facilitates capture of tail-anchored proteins by Sgt2, which interacts with tail-anchors and hands them over to the targeting component Get3. Get4/5 binds directly and with high affinity to ribosomes, positions Sgt2 close to the ribosomal tunnel exit, and facilitates the capture of tail-anchored proteins by Sgt2. The contact sites of Get4/5 on the ribosome overlap with those of SRP, the factor mediating cotranslational ER-targeting. Exposure of internal transmembrane domains at the tunnel exit induces high-affinity ribosome binding of SRP, which in turn prevents ribosome binding of Get4/5. In this way, the position of a transmembrane domain within nascent ER-targeted proteins mediates partitioning into either the GET or SRP pathway directly at the ribosomal tunnel exit.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Samatova, Daberger, Liutkute and Rodnina.)

Translation and ribosome biogenesis in mitochondria require auxiliary factors that ensure rapid and accurate synthesis of mitochondrial proteins. Defects in translation are associated with oxidative phosphorylation deficiency and cause severe human diseases, but the exact roles of mitochondrial translation-associated factors are not known. Here we identify the functions of GTPBP6, a homolog of the bacterial ribosome-recycling factor HflX, in human mitochondria. Similarly to HflX, GTPBP6 facilitates the dissociation of ribosomes in vitro and in vivo. In contrast to HflX, GTPBP6 is also required for the assembly of mitochondrial ribosomes. GTPBP6 ablation leads to accumulation of late assembly intermediate(s) of the large ribosomal subunit containing ribosome biogenesis factors MTERF4, NSUN4, MALSU1 and the GTPases GTPBP5, GTPBP7 and GTPBP10. Our data show that GTPBP6 has a dual function acting in ribosome recycling and biogenesis. These findings contribute to our understanding of large ribosomal subunit assembly as well as ribosome recycling pathway in mitochondria., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., Competing Interests: ML, MM, ES, JE, MR No competing interests declared, (© 2020, Liutkute et al.)

Alternative ribosome-rescue factor B (ArfB) rescues ribosomes stalled on non-stop mRNAs by releasing the nascent polypeptide from the peptidyl-tRNA. By rapid kinetics we show that ArfB selects ribosomes stalled on short truncated mRNAs, rather than on longer mRNAs mimicking pausing on rare codon clusters. In combination with cryo-electron microscopy we dissect the multistep rescue pathway of ArfB, which first binds to ribosomes very rapidly regardless of the mRNA length. The selectivity for shorter mRNAs arises from the subsequent slow engagement step, as it requires longer mRNA to shift to enable ArfB binding. Engagement results in specific interactions of the ArfB C-terminal domain with the mRNA entry channel, which activates peptidyl-tRNA hydrolysis by the N-terminal domain. These data reveal how protein dynamics translate into specificity of substrate recognition and provide insights into the action of a putative rescue factor in mitochondria.

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., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Efficient translational bypassing of a 50-nt non-coding gap in a phage T4 topoisomerase subunit gene (gp60) requires several recoding signals. Here we investigate the function of the mRNA stem-loop 5' of the take-off codon, as well as the importance of ribosome loading density on the mRNA for efficient bypassing. We show that polysomes are less efficient at mediating bypassing than monosomes, both in vitro and in vivo, due to their preventing formation of a stem-loop 5' of the take-off codon and allowing greater peptidyl-tRNA drop off. A ribosome profiling analysis of phage T4-infected Escherichia coli yielded protected mRNA fragments within the normal size range derived from ribosomes stalled at the take-off codon. However, ribosomes at this position also yielded some 53-nucleotide fragments, 16 longer. These were due to protection of the nucleotides that form the 5' stem-loop. NMR shows that the 5' stem-loop is highly dynamic. The importance of different nucleotides in the 5' stem-loop is revealed by mutagenesis studies. These data highlight the significance of the 5' stem-loop for the 50-nt bypassing and further enhance appreciation of relevance of the extent of ribosome loading for recoding., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

During translation, the ribosome moves along the mRNA one codon at a time with the help of elongation factor G (EF-G). Spontaneous changes in the translational reading frame are extremely rare, yet how the precise triplet-wise step is maintained is not clear. Here, we show that the ribosome is prone to spontaneous frameshifting on mRNA slippery sequences, whereas EF-G restricts frameshifting. EF-G helps to maintain the mRNA reading frame by guiding the A-site transfer RNA during translocation due to specific interactions with the tip of EF-G domain 4. Furthermore, EF-G accelerates ribosome rearrangements that restore the ribosome's control over the codon-anticodon interaction at the end of the movement. Our data explain how the mRNA reading frame is maintained during translation., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

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.

mRNA contexts containing a 'slippery' sequence and a downstream secondary structure element stall the progression of the ribosome along the mRNA and induce its movement into the -1 reading frame. In this study we build a thermodynamic model based on Bayesian statistics to explain how -1 programmed ribosome frameshifting can work. As training sets for the model, we measured frameshifting efficiencies on 64 dnaX mRNA sequence variants in vitro and also used 21 published in vivo efficiencies. With the obtained free-energy difference between mRNA-tRNA base pairs in the 0 and -1 frames, the frameshifting efficiency of a given sequence can be reproduced and predicted from the tRNA-mRNA base pairing in the two frames. Our results further explain how modifications in the tRNA anticodon modulate frameshifting and show how the ribosome tunes the strength of the base-pair interactions.

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., (© 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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.

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Assessment of the fidelity of gene expression is crucial to understand cell homeostasis. Here we present a highly sensitive method for the systematic Quantification of Rare Amino acid Substitutions (QRAS) using absolute quantification by targeted mass spectrometry after chromatographic enrichment of peptides with missense amino acid substitutions. By analyzing incorporation of near- and non-cognate amino acids in a model protein EF-Tu, we show that most of missense errors are too rare to detect by conventional methods, such as DDA, and are estimated to be between <10-7-10-5 by QRAS. We also observe error hotspots of up to 10-3 for some types of mismatches, including the G-U mismatch. The error frequency depends on the expression level of EF-Tu and, surprisingly, the amino acid position in the protein. QRAS is not restricted to any particular miscoding event, organism, strain or model protein and is a reliable tool to analyze very rare proteogenomic events., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

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., (Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.)

GTP-binding protein 1 (GTPBP1) and GTPBP2 comprise a divergent group of translational GTPases with obscure functions, which are most closely related to eEF1A, eRF3, and Hbs1. Although recent reports implicated GTPBPs in mRNA surveillance and ribosome-associated quality control, how they perform these functions remains unknown. Here, we demonstrate that GTPBP1 possesses eEF1A-like elongation activity, delivering cognate aminoacyl-transfer RNA (aa-tRNA) to the ribosomal A site in a GTP-dependent manner. It also stimulates exosomal degradation of mRNAs in elongation complexes. The kinetics of GTPBP1-mediated elongation argues against its functioning in elongation per se but supports involvement in mRNA surveillance. Thus, GTP hydrolysis by GTPBP1 is not followed by rapid peptide bond formation, suggesting that after hydrolysis, GTPBP1 retains aa-tRNA, delaying its accommodation in the A site. In physiological settings, this would cause ribosome stalling, enabling GTPBP1 to elicit quality control programs; e.g., by recruiting the exosome. GTPBP1 can also deliver deacylated tRNA to the A site, indicating that it might function via interaction with deacylated tRNA, which accumulates during stresses. Although GTPBP2's binding to GTP was stimulated by Phe-tRNA Phe , suggesting that its function might also involve interaction with aa-tRNA, GTPBP2 lacked elongation activity and did not stimulate exosomal degradation, indicating that GTPBP1 and GTPBP2 have different functions., (© 2018 Zinoviev et al.; Published by Cold Spring Harbor Laboratory Press.)

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.