ABSTRACT Templated synthesis of proteins containing non-natural amino acids (nnAAs) promises to vastly expand the chemical space available to biological therapeutics and materials. Existing technologies limit the identity and number of nnAAs than can be incorporated into a given protein. Addressing these bottlenecks requires deeper understanding of the mechanism of messenger RNA (mRNA) templated protein synthesis and how this mechanism is perturbed by nnAAs. Here we examine the impact of both monomer backbone and side chain on formation and ribosome-utilization of the central protein synthesis substate: the ternary complex of native, aminoacylated transfer RNA (aa-tRNA), thermally unstable elongation factor (EF-Tu), and GTP. By performing ensemble and single-molecule fluorescence resonance energy transfer (FRET) measurements, we reveal the dramatic effect of monomer backbone on ternary complex formation and protein synthesis. Both the (R) and (S)-β 2 isomers of Phe disrupt ternary complex formation to levels below in vitro detection limits, while (R)- and (S)-β 3 -Phe reduce ternary complex stability by approximately one order of magnitude. Consistent with these findings, (R)- and (S)-β 2 -Phe-charged tRNAs were not utilized by the ribosome, while (R)- and (S)-β 3 -Phe stereoisomers were utilized inefficiently. The reduced affinities of both species for EF-Tu ostensibly bypassed the proofreading stage of mRNA decoding. (R)-β 3 -Phe but not (S)-β 3 -Phe also exhibited order of magnitude defects in the rate of substrate translocation after mRNA decoding, in line with defects in peptide bond formation that have been observed for D-α-Phe. We conclude from these findings that non-natural amino acids can negatively impact the translation mechanism on multiple fronts and that the bottlenecks for improvement must include consideration of the efficiency and stability of ternary complex formation.

Since the framing of the Central Dogma, it has been speculated that physically distinct ribosomes within cells may influence gene expression and cellular physiology. While heterogeneity in ribosome composition has been reported in bacteria, protozoans, fungi, zebrafish, mice and humans, its functional implications remain actively debated. Here, we review recent evidence demonstrating that expression of conserved variant ribosomal DNA (rDNA) alleles in bacteria, mice and humans renders their actively translating ribosome pool intrinsically heterogeneous at the level of ribosomal RNA (rRNA). In this context, we discuss reports that nutrient limitation-induced stress in Escherichia coli leads to changes in variant rRNA allele expression, programmatically altering transcription and cellular phenotype. We highlight that cells expressing ribosomes from distinct operons exhibit distinct drug sensitivities, which can be recapitulated in vitro and potentially rationalized by subtle perturbations in ribosome structure or in their dynamic properties. Finally, we discuss evidence that differential expression of variant rDNA alleles results in different populations of ribosome subtypes within mammalian tissues. These findings motivate further research into the impacts of rRNA heterogeneities on ribosomal function and predict that strategies targeting distinct ribosome subtypes may hold therapeutic potential.This article is part of the discussion meeting issue 'Ribosome diversity and its impact on protein synthesis, development and disease'.

In all species, ribosomes synthesize proteins by faithfully decoding messenger RNA (mRNA) nucleotide sequences using aminoacyl-tRNA substrates. Current knowledge of the decoding mechanism derives principally from studies on bacterial systems 1 . Although key features are conserved across evolution 2 , eukaryotes achieve higher-fidelity mRNA decoding than bacteria 3 . In human, changes in decoding fidelity are linked to ageing and disease and represent a potential point of therapeutic intervention in both viral and cancer treatment 4–6 . Here we combine single-molecule imaging and cryogenic electron microscopy methods to examine the molecular basis of human ribosome fidelity to reveal that the decoding mechanism is both kinetically and structurally distinct from that of bacteria. Although decoding is globally analogous in both species, the reaction coordinate of aminoacyl-tRNA movement is altered on the human ribosome and the process is an order of magnitude slower. These distinctions arise from eukaryote-specific structural elements in the human ribosome and in the elongation factor eukaryotic elongation factor 1A (eEF1A) that together coordinate faithful tRNA incorporation at each mRNA codon. The distinct nature and timing of conformational changes within the ribosome and eEF1A rationalize how increased decoding fidelity is achieved and potentially regulated in eukaryotic species.

Peptide-chain elongation during protein synthesis entails sequential aminoacyl-tRNA selection and translocation reactions that proceed rapidly (2-20 per second) and with a low error rate (around 10-3 to 10-5 at each step) over thousands of cycles1. The cadence and fidelity of ribosome transit through mRNA templates in discrete codon increments is a paradigm for movement in biological systems that must hold for diverse mRNA and tRNA substrates across domains of life. Here we use single-molecule fluorescence methods to guide the capture of structures of early translocation events on the bacterial ribosome. Our findings reveal that the bacterial GTPase elongation factor G specifically engages spontaneously achieved ribosome conformations while in an active, GTP-bound conformation to unlock and initiate peptidyl-tRNA translocation. These findings suggest that processes intrinsic to the pre-translocation ribosome complex can regulate the rate of protein synthesis, and that energy expenditure is used later in the translocation mechanism than previously proposed.

Cryo electron microscopy (cryo-EM) can produce maps of macromolecules that have resolutions that are sufficiently high that structural details such as chemical modifications, water molecules and bound metal ions can be discerned. However, those accustomed to interpreting the electron-density maps of macromolecules produced by X-ray crystallography need to be careful when assigning features such as these in cryo-EM maps because cations, for example, interact far more strongly with electrons than they do with X-rays. Using simulated electrostatic potential (ESP) maps as a tool led us to re-examine a recent cryo-EM map of the human ribosome, and we realized that some of the ESP peaks originally identified as novel groups covalently bonded to the N7, O6 or O4 atoms of several guanines, adenines or uridines, respectively, in this structure are likely to instead represent Mg2+ ions coordinated to these atoms, which provide only partial charge compensation compared with Mg2+ ions located next to phosphate groups. In addition, direct evidence is provided for a variation in the level of 2′-O ribose methylation of nucleotides in the human ribosome. ESP maps can thus help in identifying ions next to nucleotide bases, i.e. at positions that can be difficult to address in cryo-EM maps due to charge effects, which are specifically encountered in cryo-EM. This work is particularly relevant to nucleoprotein complexes and shows that it is important to consider charge effects when interpreting cryo-EM maps, thus opening possibilities for localizing charges in structures that may be relevant for enzymatic mechanisms and drug interactions.

Ribosomes undergo multiple conformational transitions during translation elongation. Here, we report the high-resolution cryoelectron microscopy (cryo-EM) structure of the human 80S ribosome in the post-decoding pre-translocation state (classical-PRE) at 3.3-Å resolution along with the rotated (hybrid-PRE) and the post-translocation states (POST). The classical-PRE state ribosome structure reveals a previously unobserved interaction between the C-terminal region of the conserved ribosomal protein uS19 and the A- and P-site tRNAs and the mRNA in the decoding site. In addition to changes in the inter-subunit bridges, analysis of different ribosomal conformations reveals the dynamic nature of this domain and suggests a role in tRNA accommodation and translocation during elongation. Furthermore, we show that disease-associated mutations in uS19 result in increased frameshifting. Together, this structure-function analysis provides mechanistic insights into the role of the uS19 C-terminal tail in the context of mammalian ribosomes.

The human 80S ribosome is the cellular nucleoprotein nanomachine in charge of protein synthesis that is profoundly affected during cancer transformation by oncogenic proteins and provides cancerous proliferating cells with proteins and therefore biomass. Indeed, cancer is associated with an increase in ribosome biogenesis and mutations in several ribosomal proteins genes are found in ribosomopathies, which are congenital diseases that display an elevated risk of cancer. Ribosomes and their biogenesis therefore represent attractive anti-cancer targets and several strategies are being developed to identify efficient and specific drugs. Homoharringtonine (HHT) is the only direct ribosome inhibitor currently used in clinics for cancer treatments, although many classical chemotherapeutic drugs also appear to impact on protein synthesis. Here we review the role of the human ribosome as a medical target in cancer, and how functional and structural analysis combined with chemical synthesis of new inhibitors can synergize. The possible existence of oncoribosomes is also discussed. The emerging idea is that targeting the human ribosome could not only allow the interference with cancer cell addiction towards protein synthesis and possibly induce their death but may also be highly valuable to decrease the levels of oncogenic proteins that display a high turnover rate (MYC, MCL1). Cryo-electron microscopy (cryo-EM) is an advanced method that allows the visualization of human ribosome complexes with factors and bound inhibitors to improve our understanding of their functioning mechanisms mode. Cryo-EM structures could greatly assist the foundation phase of a novel drug-design strategy. One goal would be to identify new specific and active molecules targeting the ribosome in cancer such as derivatives of cycloheximide, a well-known ribosome inhibitor.

[This corrects the article DOI: 10.1093/narcan/zcaa036.].

RNA N6-methyladenosine (m6A) modification is present in different RNA molecules, including protein-coding mRNAs and non-coding RNAs such as ribosomal RNAs (rRNAs). Previous studies identified m6A in both the 18S and 28S rRNAs, but the roles of these methylation events are poorly understood due to the lack of knowledge of the responsible methyltransferases. Here, we report that mammalian METTL5, a member of a highly conserved methyltransferase family, specifically methylates adenosine 1832 (A1832) in the 18S rRNA in vivo and in vitro. We identify TRMT112 as a near stoichiometric partner of METTL5 important for the enzymatic activity of METTL5. By mapping the positions of translating ribosomes (Ribo-seq), we found translation of multiple stress response-related mRNAs, including Atf4 mRNA, is selectively reduced in the Mettl5 knockout (KO) mouse B16 melanoma cells. Atf4 is a key transcription factor that mediates the Integrated Stress Response (ISR), as exemplified by the Endoplasmic Reticulum (ER) stress. Consistently, transcription of ISR effector genes is reduced in Mettl5 KO cells during ER stress, suggesting a compromised ISR. Our findings reveal a new mechanism that regulates expression of stress response genes and suggest that chemical modifications of ribosomal RNAs may play a key role in selectively impacting translation and possibly ISR.

In the version of this article originally published, the references were incorrectly re-ordered during production. The hyphen in “ N 6 -methyladenosine” in the title was also superscript. The errors have been corrected in the HTML and PDF versions of the paper.

A current bottleneck in structure determination of macromolecular complexes by cryo electron microscopy (cryo-EM) is the large amount of data needed to obtain high-resolution 3D reconstructions, including through sorting into different conformations and compositions with advanced image processing. Additionally, it may be difficult to visualize small ligands that bind in sub-stoichiometric levels. Volta phase plates (VPP) introduce a phase shift in the contrast transfer and drastically increase the contrast of the recorded low-dose cryo-EM images while preserving high frequency information. Here we present a comparative study to address the behavior of different data sets during image processing and quantify important parameters during structure refinement. The automated data collection was done from the same human ribosome sample either as a conventional defocus range dataset or with a Volta phase plate close to focus (cfVPP) or with a small defocus (dfVPP). The analysis of image processing parameters shows that dfVPP data behave more robustly during cryo-EM structure refinement because particle alignments, Euler angle assignments and 2D & 3D classifications behave more stably and converge faster. In particular, less particle images are required to reach the same resolution in the 3D reconstructions. Finally, we find that defocus range data collection is also applicable to VPP. This study shows that data processing and cryo-EM map interpretation, including atomic model refinement, are facilitated significantly by performing VPP cryo-EM, which will have an important impact on structural biology.

Chemical modifications of RNA have recently gained new attention in biological sciences. They occur notably on messenger RNA (mRNA) and ribosomal RNA (rRNA) and are important for various cellular functions, but their molecular mechanism of action is yet to be understood in detail. Ribosomes are large ribonucleoprotein assemblies, which synthesize proteins in all organisms. Human ribosomes, for example, carry more than 200 modified nucleotides, which are introduced during biogenesis. Chemically modified nucleotides may appear to be only scarcely different from canonical nucleotides, but modifications such as methylations can in fact modulate their chemical and topological properties in the RNA and alter or modulate the overall translation efficiency of the ribosomes resulting in dysfunction of the translation machinery. Recent functional analysis and high-resolution ribosome structures have revealed a large repertoire of modification sites comprising different modification types. In this review, we focus on 2′-O-methylations (2′-O-Me) and discuss the structural insights gained through our recent cryo electron microscopy (cryo-EM) high-resolution structural analysis of the human ribosome, such as their locations and their influence on the secondary and tertiary structures of human rRNAs. The detailed analysis presented here reveals that ribose conformations of the rRNA backbone differ when the 2′-OH hydroxyl position is methylated, with 3′-endo conformations being the default and the 2′-endo conformations being characteristic in that the associated base is flipped-out. We compare currently known 2′-O-Me sites in human rRNAs evaluated using RiboMethSeq and cryo-EM structural analysis and discuss their involvement in several human diseases.

N-6-Methyladenosine (m(6)A) RNA modification is present in messenger RNAs (mRNA), ribosomal RNAs (rRNA), and spliceosomal RNAs (snRNA) in humans. Although mRNA m(6)A modifications have been extensively studied and shown to play critical roles in many cellular processes, the identity of m(6)A methyltransferases for rRNAs and the function of rRNA m(6)A modifications are unknown. Here we report a new m(6)A methyltransferase, ZCCHC4, which primarily methylates human 28S rRNA and also interacts with a subset of mRNAs. ZCCHC4 knockout eliminates m 6 A4220 modification in 28S rRNA, reduces global translation, and inhibits cell proliferation. We also find that ZCCHC4 protein is overexpressed in hepatocellular carcinoma tumors, and ZCCHC4 knockout significantly reduces tumor size in a xenograft mouse model. Our results highlight the functional significance of an rRNA m(6)A modification in translation and in tumor biology.

We report the revised crystal structure of a pseudo-typed human adenovirus at 3.8-Å resolution that is consistent with the atomic models of minor proteins determined by cryo-electron microscopy. The diffraction data from multiple crystals were rescaled and merged to increase the data completeness. The densities for the minor proteins were initially identified in the phase-refined omit maps that were further improved by the phases from docked poly-alanine models to build atomic structures. While the trimeric fiber molecules are disordered due to flexibility and imposition of 5-fold symmetry, the remaining major capsid proteins hexon and penton base are clearly ordered, with the exception of hypervariable region 1 of hexons, the RGD containing loop, and the N-termini of the penton base. The exterior minor protein IX together with the interior minor proteins IIIa and VIII stabilizes the adenovirus virion. A segment of N-terminal pro-peptide of VI is found in the interior cavities of peripentonal hexons, and the rest of VI is disordered. While the triskelion substructures formed by the N-termini of IX conform to excellent quasi 3-fold symmetry, the tetrameric coiled-coils formed by the C-termini and organized in parallel and anti-parallel arrangement do not exhibit any quasi-symmetry. This observation also conveys the pitfalls of using the quasi-equivalence as validation criteria for the structural analysis of extended (non-modular) capsid proteins such as IX. Together, these results remedy certain discrepancies in the previous X-ray model in agreement with the cryo-electron microscopy models.

Apart from their antimicrobial properties, tetracyclines demonstrate clinically validated effects in the amelioration of pathological inflammation and human cancer. Delineation of the target(s) and mechanism(s) responsible for these effects, however, has remained elusive. Here, employing quantitative mass spectrometry-based proteomics, we identified human 80S ribosomes as targets of the tetracyclines Col-3 and doxycycline. We then developed in-cell click selective crosslinking with RNA sequence profiling (icCL-seq) to map binding sites for these tetracyclines on key human rRNA substructures at nucleotide resolution. Importantly, we found that structurally and phenotypically variant tetracycline analogs could chemically discriminate these rRNA binding sites. We also found that tetracyclines both subtly modify human ribosomal translation and selectively activate the cellular integrated stress response (ISR). Together, the data reveal that targeting of specific rRNA substructures, activation of the ISR, and inhibition of translation are correlated with the anti-proliferative properties of tetracyclines in human cancer cell lines.

Chemical modifications of human ribosomal RNA (rRNA) are introduced during biogenesis and have been implicated in the dysregulation of protein synthesis, as is found in cancer and other diseases. However, their role in this phenomenon is unknown. Here we visualize more than 130 individual rRNA modifications in the three-dimensional structure of the human ribosome, explaining their structural and functional roles. In addition to a small number of universally conserved sites, we identify many eukaryote- or human-specific modifications and unique sites that form an extended shell in comparison to bacterial ribosomes, and which stabilize the RNA. Several of the modifications are associated with the binding sites of three ribosome-targeting antibiotics, or are associated with degenerate states in cancer, such as keto alkylations on nucleotide bases reminiscent of specialized ribosomes. This high-resolution structure of the human 80S ribosome paves the way towards understanding the role of epigenetic rRNA modifications in human diseases and suggests new possibilities for designing selective inhibitors and therapeutic drugs.

Cryo electron microscopy (cryo-EM) historically has had a strong impact on the structural and mechanistic analysis of protein synthesis by the prokaryotic and eukaryotic ribosomes. Vice versa, studying ribosomes has helped moving forwards many methodological aspects in single particle cryo-EM, at the level of automated data collection and image processing including advanced techniques for particle sorting to address structural and compositional heterogeneity. Here we review some of the latest ribosome structures, where cryo-EM allowed gaining unprecedented insights based on 3D structure sorting with focused classification and refinement methods helping to reach local resolution levels better than 3Å. Such high-resolution features now enable the analysis of drug interactions with RNA and protein side-chains including even the visualization of chemical modifications of the ribosomal RNA. These advances represent a major breakthrough in structural biology and show the strong potential of cryo-EM beyond the ribosome field including for structure-based drug design.

The interpretation of high-resolution cryo electron microscopy \(cryo-EM) maps relies on the creation of precise atomic models that help analyzing the detailed interactions within a macromolecular complex.In the accompanying paper to this protocol, we describe the high-resolution structure of the human ribosome, which includes numerous chemical modi cations.Here we present a generally applicable protocol for atomic model building and re nement into high-resolution cryo-EM maps, which in addition includes the re nement of nucleotides and amino acids with chemical modi cations.