Translation machinery captured in motion.

Translation accuracy is one of the most critical factors for protein synthesis. It is regulated by the ribosome and its dynamic behavior, along with translation factors that direct ribosome rearrangements to make translation a uniform process. Earlier structural studies of the ribosome complex with arrested translation factors laid the foundation for an understanding of ribosome dynamics and the translation process as such. Recent technological advances in time-resolved and ensemble cryo-EM have made it possible to study translation in real time at high resolution. These methods provided a detailed view of translation in bacteria for all three phases: initiation, elongation, and termination. In this review, we focus on translation factors (in some cases GTP activation) and their ability to monitor and respond to ribosome organization to enable efficient and accurate translation. This article is categorized under: Translation > Ribosome Structure/Function Translation > Mechanisms.

Structural basis of microRNA biogenesis by Dicer-1 and its partner protein Loqs-PB.

In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM structures of Drosophila Dicer-1 that show how Dicer-1 and its partner Loqs­PB cooperate (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, and (4) following complete dicing and initial product release. Our reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer­1⋅Loqs­PB heterodimer. The Dicer-1 dsRBD and three Loqs­PB dsRBDs form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-miRNA cleavage shifts the dsRBDs and partially closes Dicer-1, which may promote product release. Our data suggest a model for how the Dicer­1⋅Loqs­PB complex affects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and product release.

Ribosome inhibition by C9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM.

Toxic dipeptide-repeat (DPR) proteins are produced from expanded G4C2 repeats in the C9ORF72 gene, the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of ≥20 repeats inhibit the ribosome's peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryogenic electron microscopy (cryo-EM) reveals that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center (PTC). Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with poly-PR and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity in C9ORF72-ALS/FTD.

Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader.

Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the Saccharomyces cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale 'crab-claw' expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC's switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.

Time-resolved cryo-EM visualizes ribosomal translocation with EF-G and GTP.

During translation, a conserved GTPase elongation factor-EF-G in bacteria or eEF2 in eukaryotes-translocates tRNA and mRNA through the ribosome. EF-G has been proposed to act as a flexible motor that propels tRNA and mRNA movement, as a rigid pawl that biases unidirectional translocation resulting from ribosome rearrangements, or by various combinations of motor- and pawl-like mechanisms. Using time-resolved cryo-EM, we visualized GTP-catalyzed translocation without inhibitors, capturing elusive structures of ribosome•EF-G intermediates at near-atomic resolution. Prior to translocation, EF-G binds near peptidyl-tRNA, while the rotated 30S subunit stabilizes the EF-G GTPase center. Reverse 30S rotation releases Pi and translocates peptidyl-tRNA and EF-G by ~20 Å. An additional 4-Å translocation initiates EF-G dissociation from a transient ribosome state with highly swiveled 30S head. The structures visualize how nearly rigid EF-G rectifies inherent and spontaneous ribosomal dynamics into tRNA-mRNA translocation, whereas GTP hydrolysis and Pi release drive EF-G dissociation.

Structural basis for +1 ribosomal frameshifting during EF-G-catalyzed translocation.

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.

ArfB can displace mRNA to rescue stalled ribosomes.

Ribosomes stalled during translation must be rescued to replenish the pool of translation-competent ribosomal subunits. Bacterial alternative rescue factor B (ArfB) releases nascent peptides from ribosomes stalled on mRNAs truncated at the A site, allowing ribosome recycling. Prior structural work revealed that ArfB recognizes such ribosomes by inserting its C-terminal α-helix into the vacant mRNA tunnel. In this work, we report that ArfB can efficiently recognize a wider range of mRNA substrates, including longer mRNAs that extend beyond the A-site codon. Single-particle cryo-EM unveils that ArfB employs two modes of function depending on the mRNA length. ArfB acts as a monomer to accommodate a shorter mRNA in the ribosomal A site. By contrast, longer mRNAs are displaced from the mRNA tunnel by more than 20 Å and are stabilized in the intersubunit space by dimeric ArfB. Uncovering distinct modes of ArfB function resolves conflicting biochemical and structural studies, and may lead to re-examination of other ribosome rescue pathways, whose functions depend on mRNA lengths.

Cryo-EM of elongating ribosome with EF-Tu•GTP elucidates tRNA proofreading.

Ribosomes accurately decode mRNA by proofreading each aminoacyl-tRNA that is delivered by the elongation factor EF-Tu1. To understand the molecular mechanism of this proofreading step it is necessary to visualize GTP-catalysed elongation, which has remained a challenge2-4. Here we use time-resolved cryogenic electron microscopy to reveal 33 ribosomal states after the delivery of aminoacyl-tRNA by EF-Tu•GTP. Instead of locking cognate tRNA upon initial recognition, the ribosomal decoding centre dynamically monitors codon-anticodon interactions before and after GTP hydrolysis. GTP hydrolysis enables the GTPase domain of EF-Tu to extend away, releasing EF-Tu from tRNA. The 30S subunit then locks cognate tRNA in the decoding centre and rotates, enabling the tRNA to bypass 50S protrusions during accommodation into the peptidyl transferase centre. By contrast, the decoding centre fails to lock near-cognate tRNA, enabling the dissociation of near-cognate tRNA both during initial selection (before GTP hydrolysis) and proofreading (after GTP hydrolysis). These findings reveal structural similarity between ribosomes in initial selection states5,6 and in proofreading states, which together govern the efficient rejection of incorrect tRNA.

Characterization of novel lectins from Burkholderia pseudomallei and Chromobacterium violaceum with seven-bladed ß-propeller fold.

Burkholderia pseudomallei and Chromobacterium violaceum are bacteria of tropical and subtropical soil and water that occasionally cause fatal infections in humans and animals. Microbial lectins mediate the adhesion of organisms to host cells, which is the first phase in the development of infection. Here we report the discovery of two novel lectins from the above-mentioned bacteria - BP39L and CV39L. The crystal structures revealed that the lectins possess a seven-bladed ß-propeller fold. Functional studies conducted on a series of oligo- and polysaccharides confirmed the preference of BP39L for mannosylated saccharides and CV39L for rather more complex polysaccharides with a monosaccharide preference for ß-l-fucose. The presented data indicate that the proteins belong to a currently unknown family of lectins.

Principles for enhancing virus capsid capacity and stability from a thermophilic virus capsid structure.

The capsids of double-stranded DNA viruses protect the viral genome from the harsh extracellular environment, while maintaining stability against the high internal pressure of packaged DNA. To elucidate how capsids maintain stability in an extreme environment, we use cryoelectron microscopy to determine the capsid structure of thermostable phage P74-26 to 2.8-Å resolution. We find P74-26 capsids exhibit an overall architecture very similar to those of other tailed bacteriophages, allowing us to directly compare structures to derive the structural basis for enhanced stability. Our structure reveals lasso-like interactions that appear to function like catch bonds. This architecture allows the capsid to expand during genome packaging, yet maintain structural stability. The P74-26 capsid has T = 7 geometry despite being twice as large as mesophilic homologs. Capsid capacity is increased with a larger, flatter major capsid protein. Given these results, we predict decreased icosahedral complexity (i.e. T ≤ 7) leads to a more stable capsid assembly.

Extensive ribosome and RF2 rearrangements during translation termination.

Protein synthesis ends when a ribosome reaches an mRNA stop codon. Release factors (RFs) decode the stop codon, hydrolyze peptidyl-tRNA to release the nascent protein, and then dissociate to allow ribosome recycling. To visualize termination by RF2, we resolved a cryo-EM ensemble of E. coli 70S•RF2 structures at up to 3.3 Å in a single sample. Five structures suggest a highly dynamic termination pathway. Upon peptidyl-tRNA hydrolysis, the CCA end of deacyl-tRNA departs from the peptidyl transferase center. The catalytic GGQ loop of RF2 is rearranged into a long ß-hairpin that plugs the peptide tunnel, biasing a nascent protein toward the ribosome exit. Ribosomal intersubunit rotation destabilizes the catalytic RF2 domain on the 50S subunit and disassembles the central intersubunit bridge B2a, resulting in RF2 departure. Our structures visualize how local rearrangements and spontaneous inter-subunit rotation poise the newly-made protein and RF2 to dissociate in preparation for ribosome recycling.

Mechanism of premature translation termination on a sense codon.

Accurate translation termination by release factors (RFs) is critical for the integrity of cellular proteomes. Premature termination on sense codons, for example, results in truncated proteins, whose accumulation could be detrimental to the cell. Nevertheless, some sense codons are prone to triggering premature termination, but the structural basis for this is unclear. To investigate premature termination, we determined a cryo-EM structure of the Escherichia coli 70S ribosome bound with RF1 in response to a UAU (Tyr) sense codon. The structure reveals that RF1 recognizes a UAU codon similarly to a UAG stop codon, suggesting that sense codons induce premature termination because they structurally mimic a stop codon. Hydrophobic interaction between the nucleobase of U3 (the third position of the UAU codon) and conserved Ile-196 in RF1 is important for misreading the UAU codon. Analyses of RNA binding in ribonucleoprotein complexes or by amino acids reveal that Ile-U packing is a frequent protein-RNA-binding motif with key functional implications. We discuss parallels with eukaryotic translation termination by the release factor eRF1.

Structure and properties of AB21, a novel Agaricus bisporus protein with structural relation to bacterial pore-forming toxins.

We report the characterization of the dimeric protein AB21 from Agaricus bisporus, one of the most commonly and widely consumed mushrooms in the world. The protein shares no significant sequence similarity with any protein of known function, and it is the first characterized member of its protein family. The coding sequence of the ab21 gene was determined and the protein was expressed in E. coli in a recombinant form. We demonstrated a high thermal and pH stability of AB21 and proved the weak affinity of the protein to divalent ions of some transition metals (nickel, zinc, cadmium, and cobalt). The reported crystallographic structure exhibits an interesting rod-like helical bundle fold with structural similarity to bacterial toxins of the ClyA superfamily. By immunostaining, we demonstrated an abundance of AB21 in the fruiting bodies of A. bisporus.

Structure of RNA polymerase bound to ribosomal 30S subunit.

In bacteria, mRNA transcription and translation are coupled to coordinate optimal gene expression and maintain genome stability. Coupling is thought to involve direct interactions between RNA polymerase (RNAP) and the translational machinery. We present cryo-EM structures of E. coli RNAP core bound to the small ribosomal 30S subunit. The complex is stable under cell-like ionic conditions, consistent with functional interaction between RNAP and the 30S subunit. The RNA exit tunnel of RNAP aligns with the Shine-Dalgarno-binding site of the 30S subunit. Ribosomal protein S1 forms a wall of the tunnel between RNAP and the 30S subunit, consistent with its role in directing mRNAs onto the ribosome. The nucleic-acid-binding cleft of RNAP samples distinct conformations, suggesting different functional states during transcription-translation coupling. The architecture of the 30S•RNAP complex provides a structural basis for co-localization of the transcriptional and translational machineries, and inform future mechanistic studies of coupled transcription and translation.

Conformational dynamics are a key factor in signaling mediated by the receiver domain of a sensor histidine kinase from Arabidopsis thaliana.

Multistep phosphorelay (MSP) cascades mediate responses to a wide spectrum of stimuli, including plant hormonal signaling, but several aspects of MSP await elucidation. Here, we provide first insight into the key step of MSP-mediated phosphotransfer in a eukaryotic system, the phosphorylation of the receiver domain of the histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1RD) from Arabidopsis thaliana We observed that the crystal structures of free, Mg2+-bound, and beryllofluoridated CKI1RD (a stable analogue of the labile phosphorylated form) were identical and similar to the active state of receiver domains of bacterial response regulators. However, the three CKI1RD variants exhibited different conformational dynamics in solution. NMR studies revealed that Mg2+ binding and beryllofluoridation alter the conformational equilibrium of the ß3-α3 loop close to the phosphorylation site. Mutations that perturbed the conformational behavior of the ß3-α3 loop while keeping the active-site aspartate intact resulted in suppression of CKI1 function. Mechanistically, homology modeling indicated that the ß3-α3 loop directly interacts with the ATP-binding site of the CKI1 histidine kinase domain. The functional relevance of the conformational dynamics observed in the ß3-α3 loop of CKI1RD was supported by a comparison with another A. thaliana histidine kinase, ETR1. In contrast to the highly dynamic ß3-α3 loop of CKI1RD, the corresponding loop of the ETR1 receiver domain (ETR1RD) exhibited little conformational exchange and adopted a different orientation in crystals. Biochemical data indicated that ETR1RD is involved in phosphorylation-independent signaling, implying a direct link between conformational behavior and the ability of eukaryotic receiver domains to participate in MSP.

Characterization of novel bangle lectin from Photorhabdus asymbiotica with dual sugar-binding specificity and its effect on host immunity.

Photorhabdus asymbiotica is one of the three recognized species of the Photorhabdus genus, which consists of gram-negative bioluminescent bacteria belonging to the family Morganellaceae. These bacteria live in a symbiotic relationship with nematodes from the genus Heterorhabditis, together forming a complex that is highly pathogenic for insects. Unlike other Photorhabdus species, which are strictly entomopathogenic, P. asymbiotica is unique in its ability to act as an emerging human pathogen. Analysis of the P. asymbiotica genome identified a novel fucose-binding lectin designated PHL with a strong sequence similarity to the recently described P. luminescens lectin PLL. Recombinant PHL exhibited high affinity for fucosylated carbohydrates and the unusual disaccharide 3,6-O-Me2-Glcß1-4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 from Mycobacterium leprae. Based on its crystal structure, PHL forms a seven-bladed ß-propeller assembling into a homo-dimer with an inter-subunit disulfide bridge. Investigating complexes with different ligands revealed the existence of two sets of binding sites per monomer-the first type prefers l-fucose and its derivatives, whereas the second type can bind d-galactose. Based on the sequence analysis, PHL could contain up to twelve binding sites per monomer. PHL was shown to interact with all types of red blood cells and insect haemocytes. Interestingly, PHL inhibited the production of reactive oxygen species induced by zymosan A in human blood and antimicrobial activity both in human blood, serum and insect haemolymph. Concurrently, PHL increased the constitutive level of oxidants in the blood and induced melanisation in haemolymph. Our results suggest that PHL might play a crucial role in the interaction of P. asymbiotica with both human and insect hosts.

Ensemble cryo-EM elucidates the mechanism of translation fidelity.

Gene translation depends on accurate decoding of mRNA, the structural mechanism of which remains poorly understood. Ribosomes decode mRNA codons by selecting cognate aminoacyl-tRNAs delivered by elongation factor Tu (EF-Tu). Here we present high-resolution structural ensembles of ribosomes with cognate or near-cognate aminoacyl-tRNAs delivered by EF-Tu. Both cognate and near-cognate tRNA anticodons explore the aminoacyl-tRNA-binding site (A site) of an open 30S subunit, while inactive EF-Tu is separated from the 50S subunit. A transient conformation of decoding-centre nucleotide G530 stabilizes the cognate codon-anticodon helix, initiating step-wise 'latching' of the decoding centre. The resulting closure of the 30S subunit docks EF-Tu at the sarcin-ricin loop of the 50S subunit, activating EF-Tu for GTP hydrolysis and enabling accommodation of the aminoacyl-tRNA. By contrast, near-cognate complexes fail to induce the G530 latch, thus favouring open 30S pre-accommodation intermediates with inactive EF-Tu. This work reveals long-sought structural differences between the pre-accommodation of cognate and near-cognate tRNAs that elucidate the mechanism of accurate decoding.

Mechanism of ribosome rescue by ArfA and RF2.

ArfA rescues ribosomes stalled on truncated mRNAs by recruiting release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2 Šresolution cryo-EM structures - determined from a single sample - of the 70S ribosome with ArfA•RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA 'senses' the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled.

A Novel Fucose-binding Lectin from Photorhabdus luminescens (PLL) with an Unusual Heptabladed ß-Propeller Tetrameric Structure.

Photorhabdus luminescens is known for its symbiosis with the entomopathogenic nematode Heterorhabditis bacteriophora and its pathogenicity toward insect larvae. A hypothetical protein from P. luminescens was identified, purified from the native source, and characterized as an l-fucose-binding lectin, named P. luminescens lectin (PLL). Glycan array and biochemical characterization data revealed PLL to be specific toward l-fucose and the disaccharide glycan 3,6-O-Me2-Glcß1-4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 PLL was discovered to be a homotetramer with an intersubunit disulfide bridge. The crystal structures of native and recombinant PLL revealed a seven-bladed ß-propeller fold creating seven putative fucose-binding sites per monomer. The crystal structure of the recombinant PLL·l-fucose complex confirmed that at least three sites were fucose-binding. Moreover, the crystal structures indicated that some of the other sites are masked either by the tetrameric nature of the lectin or by incorporation of the C terminus of the lectin into one of these sites. PLL exhibited an ability to bind to insect hemocytes and the cuticular surface of a nematode, H. bacteriophora.

The influence of Mg2+ coordination on 13C and 15N chemical shifts in CKI1RD protein domain from experiment and molecular dynamics/density functional theory calculations.

Sequence dependence of (13) C and (15) N chemical shifts in the receiver domain of CKI1 protein from Arabidopsis thaliana, CKI1RD , and its complexed form, CKI1RD •Mg(2+), was studied by means of MD/DFT calculations. MD simulations of a 20-ns production run length were performed. Nine explicitly hydrated structures of increasing complexity were explored, up to a 40-amino-acid structure. The size of the model necessary depended on the type of nucleus, the type of amino acid and its sequence neighbors, other spatially close amino acids, and the orientation of amino acid NH groups and their surface/interior position. Using models covering a 10 and a 15 Å environment of Mg(2+), a semi-quantitative agreement has been obtained between experiment and theory for the V67-I73 sequence. The influence of Mg(2+) binding was described better by the 15 Å as compared to the 10 Å model. Thirteen chemical shifts were analyzed in terms of the effect of Mg(2+) insertion and geometry preparation. The effect of geometry was significant and opposite in sign to the effect of Mg(2+) binding. The strongest individual effects were found for (15) N of D70, S74, and V68, where the electrostatics dominated; for (13) Cß of D69 and (15) N of K76, where the influences were equal, and for (13) Cα of F72 and (13) Cß of K76, where the geometry adjustment dominated. A partial correlation between dominant geometry influence and torsion angle shifts upon the coordination has been observed.