N6-methyladenosine in 5' UTR does not promote translation initiation.

The most abundant N6-methyladenosine (m6A) modification on mRNAs is installed non-stoichiometrically across transcripts, with 5' untranslated regions (5' UTRs) being the least conductive. 5' UTRs are essential for translation initiation, yet the molecular mechanisms orchestrated by m6A remain poorly understood. Here, we combined structural, biochemical, and single-molecule approaches and show that at the most common position, a single m6A does not affect translation yields, the kinetics of translation initiation complex assembly, or start codon recognition both under permissive growth and following exposure to oxidative stress. Cryoelectron microscopy (cryo-EM) structures of the late preinitiation complex reveal that m6A purine ring established stacking interactions with an arginine side chain of the initiation factor eIF2α, although with only a marginal energy contribution, as estimated computationally. These findings provide molecular insights into m6A interactions with the initiation complex and suggest that the subtle stabilization is unlikely to affect the translation dynamics under homeostatic conditions or stress.

Structures of the P. aeruginosa FleQ-FleN master regulators reveal large-scale conformational switching in motility and biofilm control.

Pseudomonas aeruginosa can cause a wide array of chronic and acute infections associated with its ability to rapidly switch between planktonic, biofilm, and dispersed lifestyles, each with a specific arsenal for bacterial survival and virulence. At the cellular level, many of the physiological transitions are orchestrated by the intracellular second messenger c-di-GMP and its receptor-effector FleQ. A bacterial enhancer binding protein, FleQ acts as a master regulator of both flagellar motility and adherence factor secretion and uses remarkably different transcription activation mechanisms depending on its dinucleotide loading state, adenosine triphosphatase (ATPase) activity, interactions with polymerase sigma (σ) factors, and complexation with a second ATPase, FleN. How the FleQ-FleN tandem can exert diverse effects through recognition of a conserved FleQ binding consensus has remained enigmatic. Here, we provide cryogenic electron microscopy (cryo-EM) structures of both c-di-GMP-bound and c-di-GMP-free FleQ-FleN complexes which deepen our understanding of the proteins' (di)nucleotide-dependent conformational switching and fine-tuned roles in gene expression regulation.

Regulation of the macrolide resistance ABC-F translation factor MsrD.

Antibiotic resistance ABC-Fs (ARE ABC-Fs) are translation factors that provide resistance against clinically important ribosome-targeting antibiotics which are proliferating among pathogens. Here, we combine genetic and structural approaches to determine the regulation of streptococcal ARE ABC-F gene msrD in response to macrolide exposure. We show that binding of cladinose-containing macrolides to the ribosome prompts insertion of the leader peptide MsrDL into a crevice of the ribosomal exit tunnel, which is conserved throughout bacteria and eukaryotes. This leads to a local rearrangement of the 23 S rRNA that prevents peptide bond formation and accommodation of release factors. The stalled ribosome obstructs the formation of a Rho-independent terminator structure that prevents msrD transcriptional attenuation. Erythromycin induction of msrD expression via MsrDL, is suppressed by ectopic expression of mrsD, but not by mutants which do not provide antibiotic resistance, showing correlation between MsrD function in antibiotic resistance and its action on this stalled complex.

Purification of Mitochondrial Ribosomal Complexes from Trypanosoma cruzi and Leishmania tarentolae for Cryo-EM Analysis.

Kinetoplastids are unicellular eukaryotic parasites responsible for human pathologies such as Chagas disease, sleeping sickness or Leishmaniasis, caused by Trypanosoma cruzi, Trypanosoma brucei, and various Leishmania spp., respectively. They harbor a single large mitochondrion that is essential for the survival of the parasite. Interestingly, most of the mitochondrial gene expression machineries and processes present significant differences from their nuclear and cytosolic counterparts. A striking example concerns their mitochondrial ribosomes, in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Here, we present a detailed protocol including the specific procedures to isolate mitochondria from two species of kinetoplastids, T. cruzi and L. tarentolae, by differential centrifugations. Then, we detail the protocol to purify mitochondrial ribosomal complexes from these two species of parasites (including ribosomal maturating complexes) by a sucrose gradient approach. Finally, we describe how to prepare cryo-electron microscopy (cryo-EM) grids from these two sorts of samples. This protocol will be useful for further studies aiming at analyzing mitochondrial translation regulation.

Stabilization of Ribosomal RNA of the Small Subunit by Spermidine in Staphylococcus aureus.

Cryo-electron microscopy is now used as a method of choice in structural biology for studying protein synthesis, a process mediated by the ribosome machinery. In order to achieve high-resolution structures using this approach, one needs to obtain homogeneous and stable samples, which requires optimization of ribosome purification in a species-dependent manner. This is especially critical for the bacterial small ribosomal subunit that tends to be unstable in the absence of ligands. Here, we report a protocol for purification of stable 30 S from the Gram-positive bacterium Staphylococcus aureus and its cryo-EM structures: in presence of spermidine at a resolution ranging between 3.4 and 3.6 Å and in its absence at 5.3 Å. Using biochemical characterization and cryo-EM, we demonstrate the importance of spermidine for stabilization of the 30 S via preserving favorable conformation of the helix 44.

Functional role and ribosomal position of the unique N-terminal region of DHX29, a factor required for initiation on structured mammalian mRNAs.

Translation initiation on structured mammalian mRNAs requires DHX29, a DExH protein that comprises a unique 534-aa-long N-terminal region (NTR) and a common catalytic DExH core. DHX29 binds to 40S subunits and possesses 40S-stimulated NTPase activity essential for its function. In the cryo-EM structure of DHX29-bound 43S preinitiation complexes, the main DHX29 density resides around the tip of helix 16 of 18S rRNA, from which it extends through a linker to the subunit interface forming an intersubunit domain next to the eIF1A binding site. Although a DExH core model can be fitted to the main density, the correlation between the remaining density and the NTR is unknown. Here, we present a model of 40S-bound DHX29, supported by directed hydroxyl radical cleavage data, showing that the intersubunit domain comprises a dsRNA-binding domain (dsRBD, aa 377-448) whereas linker corresponds to the long α-helix (aa 460-512) that follows the dsRBD. We also demonstrate that the N-terminal α-helix and the following UBA-like domain form a four-helix bundle (aa 90-166) that constitutes a previously unassigned section of the main density and resides between DHX29's C-terminal α-helix and the linker. In vitro reconstitution experiments revealed the critical and specific roles of these NTR elements for DHX29's function.

How to build a ribosome from RNA fragments in Chlamydomonas mitochondria.

Mitochondria are the powerhouse of eukaryotic cells. They possess their own gene expression machineries where highly divergent and specialized ribosomes, named hereafter mitoribosomes, translate the few essential messenger RNAs still encoded by mitochondrial genomes. Here, we present a biochemical and structural characterization of the mitoribosome in the model green alga Chlamydomonas reinhardtii, as well as a functional study of some of its specific components. Single particle cryo-electron microscopy resolves how the Chlamydomonas mitoribosome is assembled from 13 rRNA fragments encoded by separate non-contiguous gene pieces. Additional proteins, mainly OPR, PPR and mTERF helical repeat proteins, are found in Chlamydomonas mitoribosome, revealing the structure of an OPR protein in complex with its RNA binding partner. Targeted amiRNA silencing indicates that these ribosomal proteins are required for mitoribosome integrity. Finally, we use cryo-electron tomography to show that Chlamydomonas mitoribosomes are attached to the inner mitochondrial membrane via two contact points mediated by Chlamydomonas-specific proteins. Our study expands our understanding of mitoribosome diversity and the various strategies these specialized molecular machines adopt for membrane tethering.

Purification and Cryo-electron Microscopy Analysis of Plant Mitochondrial Ribosomes.

Plants make up by far the largest part of biomass on Earth. They are the primary source of food and the basis of most drugs used for medicinal purposes. Similarly to all eukaryotes, plant cells also use mitochondria for energy production. Among mitochondrial gene expression processes, translation is the least understood; although, recent advances have revealed the specificities of its main component, the mitochondrial ribosome (mitoribosome). Here, we present a detailed protocol to extract highly pure cauliflower mitochondria by differential centrifugation for the purification of mitochondrial ribosomes using a sucrose gradient and the preparation of cryo-electron microscopy (cryo-EM) grids. Finally, the specific bioinformatics pipeline used for image acquisition, the processing steps, and the data analysis used for cryo-EM of the plant mitoribosome are described. This protocol will be used for further analysis of the critical steps of mitochondrial translation, such as its initiation and regulation.

Peculiarities of aminoacyl-tRNA synthetases from trypanosomatids.

Trypanosomatid parasites are responsible for various human diseases, such as sleeping sickness, animal trypanosomiasis, or cutaneous and visceral leishmaniases. The few available drugs to fight related parasitic infections are often toxic and present poor efficiency and specificity, and thus, finding new molecular targets is imperative. Aminoacyl-tRNA synthetases (aaRSs) are essential components of the translational machinery as they catalyze the specific attachment of an amino acid onto cognate tRNA(s). In trypanosomatids, one gene encodes both cytosolic- and mitochondrial-targeted aaRSs, with only three exceptions. We identify here a unique specific feature of aaRSs from trypanosomatids, which is that most of them harbor distinct insertion and/or extension sequences. Among the 26 identified aaRSs in the trypanosome Leishmania tarentolae, 14 contain an additional domain or a terminal extension, confirmed in mature mRNAs by direct cDNA nanopore sequencing. Moreover, these RNA-Seq data led us to address the question of aaRS dual localization and to determine splice-site locations and the 5'-UTR lengths for each mature aaRS-encoding mRNA. Altogether, our results provided evidence for at least one specific mechanism responsible for mitochondrial addressing of some L. tarentolae aaRSs. We propose that these newly identified features of trypanosomatid aaRSs could be developed as relevant drug targets to combat the diseases caused by these parasites.

Interaction Networks of Ribosomal Expansion Segments in Kinetoplastids.

Expansion segments (ES) are insertions of a few to hundreds of nucleotides at discrete locations on eukaryotic ribosomal RNA (rRNA) chains. Some cluster around 'hot spots' involved in translation regulation and some may participate in biogenesis. Whether ES play the same roles in different organisms is currently unclear, especially since their size may vary dramatically from one species to another and very little is known about their functions. Most likely, ES variation is linked to adaptation to a particular environment. In this chapter, we compare the interaction networks of ES from four kinetoplastid parasites, which have evolved in diverse insect vectors and mammalian hosts: Trypanosoma cruzi, Trypanosoma brucei, Leishmania donovani and Leishmania major. Here, we comparatively analyze ribosome structures from these representative kinetoplastids and ascertain meaningful structural differences from mammalian ribosomes. We base our analysis on sequence alignments and three-dimensional structures of 80S ribosomes solved by cryo-electron microscopy (cryo-EM). Striking differences in size are observed between ribosomes of different parasites, indicating that not all ES are expanded equally. Larger ES are not always matched by large surrounding ES or proteins extensions in their vicinity, a particularity that may lead to clues about their biological function. ES display different species-specific patterns of conservation, which underscore the density of their interaction network at the surface of the ribosome. Making sense of the conservation patterns of ES is part of a global effort to lay the basis for functional studies aimed at discovering unique kinetoplastid-specific sites suitable for therapeutic applications against these human and often animal pathogens.

The Halastavi árva Virus Intergenic Region IRES Promotes Translation by the Simplest Possible Initiation Mechanism.

Dicistrovirus intergenic region internal ribosomal entry sites (IGR IRESs) do not require initiator tRNA, an AUG codon, or initiation factors and jumpstart translation from the middle of the elongation cycle via formation of IRES/80S complexes resembling the pre-translocation state. eEF2 then translocates the [codon-anticodon]-mimicking pseudoknot I (PKI) from ribosomal A sites to P sites, bringing the first sense codon into the decoding center. Halastavi árva virus (HalV) contains an IGR that is related to previously described IGR IRESs but lacks domain 2, which enables these IRESs to bind to individual 40S ribosomal subunits. By using in vitro reconstitution and cryoelectron microscopy (cryo-EM), we now report that the HalV IGR IRES functions by the simplest initiation mechanism that involves binding to 80S ribosomes such that PKI is placed in the P site, so that the A site contains the first codon that is directly accessible for decoding without prior eEF2-mediated translocation of PKI.

Structural Differences in Translation Initiation between Pathogenic Trypanosomatids and Their Mammalian Hosts.

Canonical mRNA translation in eukaryotes begins with the formation of the 43S pre-initiation complex (PIC). Its assembly requires binding of initiator Met-tRNAiMet and several eukaryotic initiation factors (eIFs) to the small ribosomal subunit (40S). Compared to their mammalian hosts, trypanosomatids present significant structural differences in their 40S, suggesting substantial variability in translation initiation. Here, we determine the structure of the 43S PIC from Trypanosoma cruzi, the parasite causing Chagas disease. Our structure shows numerous specific features, such as the variant eIF3 structure and its unique interactions with the large rRNA expansion segments (ESs) 9S, 7S, and 6S, and the association of a kinetoplastid-specific DDX60-like helicase. It also reveals the 40S-binding site of the eIF5 C-terminal domain and structures of key terminal tails of several conserved eIFs underlying their activities within the PIC. Our results are corroborated by glutathione S-transferase (GST) pull-down assays in both human and T. cruzi and mass spectrometry data.

Structure of the mature kinetoplastids mitoribosome and insights into its large subunit biogenesis.

Kinetoplastids are unicellular eukaryotic parasites responsible for such human pathologies as Chagas disease, sleeping sickness, and leishmaniasis. They have a single large mitochondrion, essential for the parasite survival. In kinetoplastid mitochondria, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Structural studies performed in Trypanosoma brucei already highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit. However, several important aspects mainly related to the large subunit (LSU) remain elusive, such as the structure and maturation of its ribosomal RNA. Here we present a cryo-electron microscopy study of the protozoans Leishmania tarentolae and Trypanosoma cruzi mitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the LSU, as well as previously unidentified ribosomal proteins. In addition, we introduce the structure of an LSU assembly intermediate in the presence of 16 identified maturation factors. These maturation factors act on both the intersubunit and the solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.

Specific features and assembly of the plant mitochondrial complex I revealed by cryo-EM.

Mitochondria are the powerhouses of eukaryotic cells and the site of essential metabolic reactions. Complex I or NADH:ubiquinone oxidoreductase is the main entry site for electrons into the mitochondrial respiratory chain and constitutes the largest of the respiratory complexes. Its structure and composition vary across eukaryote species. However, high resolution structures are available only for one group of eukaryotes, opisthokonts. In plants, only biochemical studies were carried out, already hinting at the peculiar composition of complex I in the green lineage. Here, we report several cryo-electron microscopy structures of the plant mitochondrial complex I. We describe the structure and composition of the plant respiratory complex I, including the ancestral mitochondrial domain composed of the carbonic anhydrase. We show that the carbonic anhydrase is a heterotrimeric complex with only one conserved active site. This domain is crucial for the overall stability of complex I as well as a peculiar lipid complex composed of cardiolipin and phosphatidylinositols. Moreover, we also describe the structure of one of the plant-specific complex I assembly intermediates, lacking the whole PD module, in presence of the maturation factor GLDH. GLDH prevents the binding of the plant specific P1 protein, responsible for the linkage of the PP to the PD module.

Escherichia coli NusG Links the Lead Ribosome with the Transcription Elongation Complex.

It has been known for more than 50 years that transcription and translation are physically coupled in bacteria, but whether or not this coupling may be mediated by the two-domain protein N-utilization substance (Nus) G in Escherichia coli is still heavily debated. Here, we combine integrative structural biology and functional analyses to provide conclusive evidence that NusG can physically link transcription with translation by contacting both RNA polymerase and the ribosome. We present a cryo-electron microscopy structure of a NusG:70S ribosome complex and nuclear magnetic resonance spectroscopy data revealing simultaneous binding of NusG to RNAP and the intact 70S ribosome, providing the first direct structural evidence for NusG-mediated coupling. Furthermore, in vivo reporter assays show that recruitment of NusG occurs late in transcription and strongly depends on translation. Thus, our data suggest that coupling occurs initially via direct RNAP:ribosome contacts and is then mediated by NusG.

Cryo-EM structure of the RNA-rich plant mitochondrial ribosome.

The vast majority of eukaryotic cells contain mitochondria, essential powerhouses and metabolic hubs1. These organelles have a bacterial origin and were acquired during an early endosymbiosis event2. Mitochondria possess specialized gene expression systems composed of various molecular machines, including the mitochondrial ribosomes (mitoribosomes). Mitoribosomes are in charge of translating the few essential mRNAs still encoded by mitochondrial genomes3. While chloroplast ribosomes strongly resemble those of bacteria4,5, mitoribosomes have diverged significantly during evolution and present strikingly different structures across eukaryotic species6-10. In contrast to animals and trypanosomatids, plant mitoribosomes have unusually expanded ribosomal RNAs and have conserved the short 5S rRNA, which is usually missing in mitoribosomes11. We have previously characterized the composition of the plant mitoribosome6, revealing a dozen plant-specific proteins in addition to the common conserved mitoribosomal proteins. In spite of the tremendous recent advances in the field, plant mitoribosomes remained elusive to high-resolution structural investigations and the plant-specific ribosomal features of unknown structures. Here, we present a cryo-electron microscopy study of the plant 78S mitoribosome from cauliflower at near-atomic resolution. We show that most of the plant-specific ribosomal proteins are pentatricopeptide repeat proteins (PPRs) that deeply interact with the plant-specific rRNA expansion segments. These additional rRNA segments and proteins reshape the overall structure of the plant mitochondrial ribosome, and we discuss their involvement in the membrane association and mRNA recruitment prior to translation initiation. Finally, our structure unveils an rRNA-constructive phase of mitoribosome evolution across eukaryotes.

Specificities of the plant mitochondrial translation apparatus.

Mitochondria are endosymbiotic organelles responsible for energy production in most eukaryotic cells. They host a genome and a fully functional gene expression machinery. In plants this machinery involves hundreds of pentatricopeptide repeat (PPR) proteins. Translation, the final step of mitochondrial gene expression is performed by mitochondrial ribosomes (mitoribosomes). The nature of these molecular machines remained elusive for a very long time. Because of their bacterial origin, it was expected that mitoribosomes would closely resemble bacterial ribosomes. However, recent advances in cryo-electron microscopy have revealed the extraordinary diversity of mitoribosome structure and composition. The plant mitoribosome was characterized for Arabidopsis. In plants, in contrast to other species such as mammals and kinetoplastids where rRNA has been largely reduced, the mitoribosome could be described as a protein/RNA-augmented bacterial ribosome. It has an oversized small subunit formed by expanded ribosomal RNAs and additional protein components when compared to bacterial ribosomes. The same holds true for the large subunit. The small subunit is characterized by a new elongated domain on the head. Among its additional proteins, several PPR proteins are core mitoribosome proteins. They mainly act at the structural level to stabilize and maintain the plant-specific ribosomal RNA expansions but could also be involved in translation initiation. Recent advances in plant mitoribosome composition and structure, its specialization for membrane protein synthesis, translation initiation, the regulation and dynamics of mitochondrial translation are reviewed here and put in perspective with the diversity of mitochondrial translation processes in the green lineage and in the wider context of eukaryote evolution.

Structural Insights into the Mammalian Late-Stage Initiation Complexes.

In higher eukaryotes, the mRNA sequence in the direct vicinity of the start codon, called the Kozak sequence (CRCCaugG, where R is a purine), is known to influence the rate of the initiation process. However, the molecular basis underlying its role remains poorly understood. Here, we present the cryoelectron microscopy (cryo-EM) structures of mammalian late-stage 48S initiation complexes (LS48S ICs) in the presence of two different native mRNA sequences, ß-globin and histone 4, at overall resolution of 3 and 3.5 Å, respectively. Our high-resolution structures unravel key interactions from the mRNA to eukaryotic initiation factors (eIFs): 1A, 2, 3, 18S rRNA, and several 40S ribosomal proteins. In addition, we are able to study the structural role of ABCE1 in the formation of native 48S ICs. Our results reveal a comprehensive map of ribosome/eIF-mRNA and ribosome/eIF-tRNA interactions and suggest the impact of mRNA sequence on the structure of the LS48S IC.

Small is big in Arabidopsis mitochondrial ribosome.

Mitochondria are responsible for energy production through aerobic respiration, and represent the powerhouse of eukaryotic cells. Their metabolism and gene expression processes combine bacterial-like features and traits that evolved in eukaryotes. Among mitochondrial gene expression processes, translation remains the most elusive. In plants, while numerous pentatricopeptide repeat (PPR) proteins are involved in all steps of gene expression, their function in mitochondrial translation remains unclear. Here we present the biochemical characterization of Arabidopsis mitochondrial ribosomes and identify their protein subunit composition. Complementary biochemical approaches identified 19 plant-specific mitoribosome proteins, of which ten are PPR proteins. The knockout mutations of ribosomal PPR (rPPR) genes result in distinct macroscopic phenotypes, including lethality and severe growth delay. The molecular analysis of rppr1 mutants using ribosome profiling, as well as the analysis of mitochondrial protein levels, demonstrate rPPR1 to be a generic translation factor that is a novel function for PPR proteins. Finally, single-particle cryo-electron microscopy (cryo-EM) reveals the unique structural architecture of Arabidopsis mitoribosomes, characterized by a very large small ribosomal subunit, larger than the large subunit, bearing an additional RNA domain grafted onto the head. Overall, our results show that Arabidopsis mitoribosomes are substantially divergent from bacterial and other eukaryote mitoribosomes, in terms of both structure and protein content.