Search

The ribosome serves as the universally conserved translator of the genetic code into proteins and supports life across diverse temperatures ranging from below freezing to above 120°C. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3′-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not affect cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation across archaeal ribosomes in the peptidyl transferase center that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.

An anaerobic microbial enrichment culture was used to study members of candidate phyla that are difficult to grow in the lab. We were able to visualize tiny “ Candidatus Nealsonbacteria” cells attached to a large Methanothrix cell, revealing a novel episymbiosis.

Cell-free protein synthesis (CFPS) systems enable easyin vitroexpression of proteins with many scientific, industrial, and therapeutic applications. Here we present an optimized, highly efficient human cell-free translation system that bypasses many limitations of currently usedin vitrosystems. This CFPS system is based on extracts from human HEK293T cells engineered to endogenously express GADD34 and K3L proteins, which suppress phosphorylation of translation initiation factor eIF2α. Overexpression of GADD34 and K3L proteins in human cells significantly simplifies cell lysate preparation. The new CFPS system improves the translation of 5’ cap-dependent mRNAs as well as those that use internal ribosome entry site (IRES) mediated translation initiation. We find that expression of the GADD34 and K3L accessory proteins before cell lysis maintains low levels of phosphorylation of eIF2α in the extracts. Duringin vitrotranslation reactions, eIF2α phosphorylation increases moderately in a GCN2-dependent fashion that can be inhibited by GCN2 kinase inhibitors. We also find evidence for activation of regulatory pathways related to eukaryotic elongation factor 2 (eEF2) phosphorylation and ribosome quality control in the extracts. This new CFPS system should be useful for exploring human translation mechanisms in more physiological conditions outside the cell.

TheEscherichia coliribosome can incorporate a variety of non-l-α-amino acid monomers into polypeptide chains, but with poor efficiency. Although these monomers span a diverse set of compounds, there exists no high-resolution structural information regarding their positioning within the catalytic center of the ribosome, the peptidyl transferase center (PTC). Thus, details regarding the mechanism of amide bond formation and the structural basis for differences and defects in incorporation efficiency remain unknown. Within a set of three aminobenzoic acid derivatives–3-aminopyridine-4-carboxylic acid (Apy),ortho-aminobenzoic acid (oABZ), andmeta-aminobenzoic acid (mABZ)–the ribosome incorporates Apy into polypeptide chains with the highest efficiency, followed byoABZ and thenmABZ, a trend that does not track with the nucleophilicity of the reactive amines. Here, we report high resolution cryo-EM structures of the ribosome with these three aminobenzoic acid derivatives charged on tRNA bound in the aminoacyl-tRNA site (A site). These structures reveal how the aromatic ring of each monomer sterically blocks positioning of nucleotide U2506, thereby preventing rearrangement of nucleotide U2585 and the resulting induced fit in the PTC required for efficient amide bond formation. They also reveal disruptions to the “proton wire” responsible for facilitating formation and breakdown of the tetrahedral intermediate. Together, the cryo-EM structures reported here provide a clear rationale for differences in reactivity of aminobenzoic acid derivatives relative tol-α-amino acids and each other, and point to stereochemical constraints on the size and geometry of non-proteinogenic monomers that can be accepted efficiently by wild-type ribosomes.

The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding.In vivoassembled ribosomes have been isolated using MS2-tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and functionin vitro. RNA tags in theEscherichia coli50S subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth orin vitroribosome activity. Here, we find thatE. coli50S subunits with MS2 tags inserted in H98 are destabilized compared to wild type (WT) 50S subunits. We identify the loss of RNA-RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes.

As genetic code expansion advances beyond L-α-amino acids to backbone modifications and new polymerization chemistries, the field faces an increasingly broad challenge to discover what the ribosome can accommodate. Although the E. coli ribosome tolerates non-L-α-amino acids in vitro, few structural insights are available, and the boundary conditions for efficient bond formation are unknown. We describe a 2.1 Å cryo-EM structure of the E. coli ribosome containing well-resolved α-amino acid monomers coupled with a computational approach for which energy surface minima produced by metadynamics trend in agreement with established incorporation efficiencies. Reactive monomers across diverse structural classes favor a conformational space characterized by an A-site nucleophile to P-site carbonyl distance of < 4 Å and a Bürgi-Dunitz angle of 90-110°. Monomers whose free energy minima fall outside these regions do not react. Application of this model should accelerate the in vivo and in vitro ribosomal synthesis and application of sequence-defined, non-peptide heterooligomers.

Ribosomes serve as the universally conserved translators of the genetic code into proteins and must support life across temperatures ranging from below freezing to above the boiling point of water. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Peptide bond formation by the ribosome requires correct positioning of the 3’ s-end of the aminoacylated tRNA (aa-tRNA) substrate, which is aided by an RNA hairpin in the ribosomal RNA (rRNA) of the large subunit, termed the A loop. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3′-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not exert a dominant negative effect on cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation in the peptidyl transferase center of the archaeal ribosome that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.

Candidate phyla radiation (CPR) bacteria and DPANN archaea are unisolated, small-celled symbionts that are often detected in groundwater. The effects of groundwater geochemistry on the abundance, distribution, taxonomic diversity and host association of CPR bacteria and DPANN archaea has not been studied. Here, we performed genome-resolved metagenomic analysis of one agricultural and seven pristine groundwater microbial communities and recovered 746 CPR and DPANN genomes in total. The pristine sites, which serve as local sources of drinking water, contained up to 31% CPR bacteria and 4% DPANN archaea. We observed little species-level overlap of metagenome-assembled genomes (MAGs) across the groundwater sites, indicating that CPR and DPANN communities may be differentiated according to physicochemical conditions and host populations. Cryogenic transmission electron microscopy imaging and genomic analyses enabled us to identify CPR and DPANN lineages that reproducibly attach to host cells and showed that the growth of CPR bacteria seems to be stimulated by attachment to host-cell surfaces. Our analysis reveals site-specific diversity of CPR bacteria and DPANN archaea that coexist with diverse hosts in groundwater aquifers. Given that CPR and DPANN organisms have been identified in human microbiomes and their presence is correlated with diseases such as periodontitis, our findings are relevant to considerations of drinking water quality and human health., Metagenomics and electron microscopy are combined to analyse the diversity of episymbiotic CPR bacteria and DPANN archaea in eight groundwater communities.

SummaryThe Candidate Phyla Radiation (CPR) is a very large group of bacteria with no pure culture representatives, first discovered by metagenomic analyses. Within the CPR, candidate phylum Parcubacteria (previously referred to as OD1) within the candidate superphylum Patescibacteria is prevalent in anoxic sediments and groundwater. Previously, we had identified a specific member of the Parcubacteria (referred to as DGGOD1a) as an important member of a methanogenic benzene-degrading consortium. Phylogenetic analyses herein place DGGOD1a within theCandidateclade Nealsonbacteria. Because of its persistence over many years, we hypothesized thatCa. Nealsonbacteria DGGOD1a must serve an important role in sustaining anaerobic benzene metabolism in the consortium. To try to identify its growth substrate, we amended the culture with a variety of defined compounds (pyruvate, acetate, hydrogen, DNA, phospholipid), as well as crude culture lysate and three subfractions thereof. We observed the greatest (10 fold) increase in the absolute abundance ofCa. Nealsonbacteria DGGOD1a only when the consortium was amended with crude cell lysate. These results implicateCa. Nealsonbacteria in biomass recycling. Fluorescent in situ hybridization and cryogenic transmission electron microscope images revealed thatCa. Nealsonbacteria DGGOD1a cells were attached to larger archaealMethanothrixcells. This apparent epibiont lifestyle was supported by metabolic predictions from a manually curated complete genome. This is one of the first examples of bacterial-archaeal episymbiosis and may be a feature of otherCa. Nealsonbacteria found in anoxic environments.

SummaryThe advancement of precision engineering for crop trait improvement is important in the face of rapid population growth, climate change, and disease. To this end, targeted double-stranded break technology using RNA-guided Cas9 has been adopted widely for genome editing in plants. Agrobacterium or particle bombardment-based delivery of plasmids encoding Cas9 and guide RNA (gRNA) is common, but requires optimization of expression and often results in random integration of plasmid DNA into the plant genome. Recent advances have described gene editing by the delivery of Cas9 and gRNA as pre-assembled ribonucleoproteins (RNPs) into various plant tissues, but with moderate efficiency in resulting regenerated plants. In this report we describe significant improvements to Cas9-RNP mediated gene editing in wheat. We demonstrate that Cas9-RNP assays in protoplasts are a fast and effective tool for rational selection of optimal gRNAs for gene editing in regenerable immature embryos (IEs), and that high temperature treatment enhances gene editing rates in both tissue types. We also show that Cas9-mediated editing persists for at least 14 days in gold particle bombarded wheat IEs. The regenerated edited wheat plants in this work are recovered at high rates in the absence of exogenous DNA and selection. With this method, we produce knockouts of a set of three homoeologous genes and two pathogenic effector susceptibility genes that result in insensitivity to corresponding necrotrophic effectors produced by Parastagonospora nodorum. The establishment of highly efficient, DNA-free gene editing technology holds promise for accelerated trait diversity production in an expansive array of crops.

Methods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation. These molecules also inhibit translation termination by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 inhibits translation termination by arresting the nascent chain (NC) in the ribosome exit tunnel. The arrested NC adopts a compact α-helical conformation that induces 28 S rRNA nucleotide rearrangements that suppress the peptidyl transferase center (PTC) catalytic activity stimulated by eukaryotic release factor 1 (eRF1). These data support a mechanism of action for a small molecule targeting translation that suppresses peptidyl-tRNA hydrolysis promoted by eRF1, revealing principles of eukaryotic translation termination and laying the foundation for new therapeutic strategies., The drug-like compound PF846 and its derivatives inhibit the translation of specific mRNAs by the human ribosome. Here the authors show how PF846 arrests translation at the stop codon by slowing hydrolysis of the protein nascent chain at the ribosome P-site tRNA by eukaryotic release factor 1 (eRF1).

Activation of T cells requires a rapid surge in cellular protein synthesis. However, the role of translation initiation in the early induction of specific genes remains unclear. Here we show human translation initiation factor eIF3 interacts with select immune system related mRNAs including those encoding the T cell receptor (TCR) subunits TCRA and TCRB. Binding of eIF3 to theTCRAandTCRBmRNA 3’-untranslated regions (3’-UTRs) depends on CD28 coreceptor signaling and regulates a burst in TCR translation required for robust T cell activation. Use of theTCRAorTCRB3’-UTRs to control expression of an anti-CD19 chimeric antigen receptor (CAR) improves the ability of CAR-T cells to kill tumor cellsin vitro. These results identify a new mechanism of eIF3-mediated translation control that can aid T cell engineering for immunotherapy applications.

Nonstructural protein 1 (nsp1) is a coronavirus (CoV) virulence factor that restricts cellular gene expression by inhibiting translation through blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We perform a detailed structure-guided mutational analysis of severe acute respiratory syndrome (SARS)-CoV-2 nsp1, revealing insights into how it coordinates these activities against host but not viral mRNA. We find that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, both enhancing its restriction of host gene expression and enabling mRNA containing the SARS-CoV-2 leader sequence to escape translational repression. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in SARS-CoV-2 nsp1 could attenuate the virus., Graphical abstract, The coronavirus Nsp1 protein binds the 40S ribosomal subunit to induce host translational suppression and mRNA cleavage. Mendez et al. reveal that mutation of specific residues in the Nsp1 N terminus destabilizes its interaction with the 40S subunit and eliminates the protection of viral-leader-sequence-containing transcripts from translational repression.

Ribosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant ribosomes have been developed that improve the incorporation of several non-standard monomers including D-amino acids, dipeptides, and β-amino acids into polypeptide chains. However, there remains little mechanistic understanding of how these ribosomes catalyze incorporation of these new substrates. Here we probed the properties of a mutant ribosome–P7A7–evolved for betterin vivoβ-amino acid incorporation throughin vitrobiochemistry and cryo-electron microscopy. Although P7A7 is a functional ribosomein vivo, it is inactivein vitro, and assembles poorly into 70S complexes. Structural characterization revealed large regions of disorder in the peptidyltransferase center and nearby features, suggesting a defect in assembly. Comparison of RNA helix and ribosomal protein occupancy with other assembly intermediates revealed that P7A7 is stalled at a late stage in ribosome assembly, explaining its weak activity. These results highlight the importance of ensuring efficient ribosome assembly during ribosome engineering towards new catalytic abilities.

Small molecules that target the ribosome generally have a global impact on protein synthesis. However, the drug-like molecule PF-06446846 (PF846) binds the human ribosome and selectively blocks the translation of a small subset of proteins by an unknown mechanism. In high-resolution cryo-electron microscopy (cryo-EM) structures of human ribosome nascent chain complexes stalled by PF846, PF846 binds in the ribosome exit tunnel in a newly-identified and eukaryotic-specific pocket formed by the 28S ribosomal RNA (rRNA), and redirects the path of the nascent polypeptide chain. PF846 arrests the translating ribosome in the rotated state that precedes mRNA and tRNA translocation, with peptidyl-tRNA occupying a mixture of A/A and hybrid A/P sites, in which the tRNA 3’-CCA end is improperly docked in the peptidyl transferase center. Using mRNA libraries, selections of PF846-dependent translation elongation stalling sequences reveal sequence preferences near the peptidyl transferase center, and uncover a newly-identified mechanism by which PF846 selectively blocks translation termination. These results illuminate how a small molecule selectively stalls the translation of the human ribosome, and provides a structural foundation for developing small molecules that inhibit the production of proteins of therapeutic interest.

The ribosome exit tunnel is an important structure involved in the regulation of translation and other essential functions such as protein folding. By comparing 20 recently obtained cryo-EM and X-ray crystallography structures of the ribosome from all three domains of life, we here characterize the key similarities and differences of the tunnel across species. We first show that a hierarchical clustering of tunnel shapes closely reflects the species phylogeny. Then, by analyzing the ribosomal RNAs and proteins, we explain the observed geometric variations and show direct association between the conservations of the geometry, structure and sequence. We find that the tunnel is more conserved in the upper part close to the polypeptide transferase center, while in the lower part, it is substantially narrower in eukaryotes than in bacteria. Furthermore, we provide evidence for the existence of a second constriction site in eukaryotic exit tunnels. Overall, these results have several evolutionary and functional implications, which explain certain differences between eukaryotes and prokaryotes in their translation mechanisms. In particular, they suggest that major co-translational functions of bacterial tunnels were externalized in eukaryotes, while reducing the tunnel size provided some other advantages, such as facilitating the nascent chain elongation and enabling antibiotic resistance.

Ribosomes consist of many small proteins and few large RNA molecules. Both components are necessary for ribosome functioning during translation. According to widely accepted view, bacterial ribosomes contain always the same complement of ribosomal proteins. Comparative bacterial genomics data indicates that several ribosomal proteins are encoded by multiple paralogous genes suggesting structural heterogeneity of ribosomes. In E. coli, two r-proteins bL31 and bL36 are encoded by two genes: rpmE and ykgM encode bL31 protein paralogs bL31A and bL31B, and rpmJ and ykgO encode bL36 protein paralogs bL36A and bL36B respectively. We have found several similarities and differences between ribosomes of exponential and stationary growth phases by using quantitative mass spectrometry and X-ray crystallography. First, composition of ribosome associating proteins changes profoundly as cells transition from exponential to stationary growth phase. Ribosomal core proteins bL31A and bL36A are replaced by bL31B and bL36B, respectively. Second, our X-ray structure of the 70S ribosome demonstrates that bL31B and bL36B proteins have similar ribosome binding sites to their A counterparts. Third, ribosome subpopulations containing A or B paralogs existed simultaneously demonstrating that E. coli ribosomes are heterogeneous with respect to their paralogous ribosomal protein composition that changes via protein exchange.

Nonstructural protein 1 (nsp1) is the first viral protein synthesized during coronavirus (CoV) infection and is a key virulence factor that dampens the innate immune response. It restricts cellular gene expression through a combination of inhibiting translation by blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We performed a detailed structure-guided mutational analysis of CoV-2 nsp1 coupled with in vitro and cell-based functional assays, revealing insight into how it coordinates these activities against host but not viral mRNA. We found that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, thereby enhancing its restriction of host gene expression. These residues are also critical for the ability of mRNA containing the CoV-2 leader sequence to escape translational repression. Notably, we identify CoV-2 nsp1 mutants that gain the ability to repress translation of viral leader-containing transcripts. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in CoV-2 nsp1 could attenuate the virus.

Nonstructural protein 1 (nsp1) is the first viral protein synthesized during coronavirus (CoV) infection and is a key virulence factor that dampens the innate immune response. It restricts cellular gene expression through a combination of inhibiting translation by blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We performed a detailed structure-guided mutational analysis of CoV-2 nsp1 coupled with in vitro and cell-based functional assays, revealing insight into how it coordinates these activities against host but not viral mRNA. We found that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, thereby enhancing its restriction of host gene expression. These residues are also critical for the ability of mRNA containing the CoV-2 leader sequence to escape translational repression. Notably, we identify CoV-2 nsp1 mutants that gain the ability to repress translation of viral leader-containing transcripts. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in CoV-2 nsp1 could attenuate the virus. Funding Information: This project was funded by a COVID19 Excellence in Research Award from the Laboratory of Genomics Research to BAG, JHC and NTI and an Emergent Ventures COVID19 Fast Grant #2155 to BAG, who is also an investigator of the Howard Hughes Medical Institute. ML is funded by NSERC Predoctoral Fellowship PGSD3-516787-2018. Declaration of Interests: No competing interests to declare. Ethics Approval Statement: N/A; no animal or human subjects work in this manuscript.

Background An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day. Results Here we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo. Conclusion The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.

Continuing advances in cryo-electron microscopy (cryo-EM) demonstrate the promise it holds for revealing biological structures at chemical resolution, in which noncovalent interactions, RNA and protein modifications, and solvation can be modeled accurately. At present, the best cryo-EM-derived models of the bacterial ribosome are of the large (50S) ribosomal subunit with effective global resolutions of 2.4-2.5 Å, based on map-to-model Fourier shell correlation (FSC). Here we present a model of theE. coli70S ribosome with an effective global resolution of 2.0 Å, based on maps showcasing unambiguous positioning of residues, their detailed chemical interactions, and chemical modifications. These modifications include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former of which is likely conserved in all domains of life. The model also defines extensive solvation of the small (30S) ribosomal subunit for the first time, as well as interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The high quality of the maps now allows a deeper phylogenetic analysis of ribosomal components, and identification of structural conservation to the level of solvation. The maps and models of the bacterial ribosome presented here should enable future structural analysis of the chemical basis for translation, and the development of robust tools for cryo-EM structure modeling and refinement.

Candidate Phyla Radiation (CPR) bacteria and DPANN archaea are uncultivated, small-celled symbionts often detected in groundwater. However, variations in CPR/DPANN organism abundance, distribution, taxonomic diversity, and degree/nature of host association with groundwater chemistry remain understudied. Here, we performed genome-resolved metagenomic characterization of one agriculturally-impacted and seven pristine groundwater microbial communities in California, recovering 746 dereplicated CPR and DPANN genomes. Our finding of up to 31% CPR bacteria and 4% DPANN archaea in the pristine sites, which serve as local sources of drinking water, may hold health relevance, given growing awareness of the presence of CPR/DPANN organisms in human microbiomes and their association with disease. There is little species-level genome overlap across groundwater sites, indicating that CPR and DPANN communities are highly differentiated according to host populations and physicochemical conditions. Cryo-TEM imaging and genomic analyses indicate that CPR growth may be stimulated by attachment to the surface of host cells, and identified CPR and DPANN lineages with particularly prevalent and/or resilient host cell attachment. These results establish the huge but site-specific diversity of CPR bacteria and DPANN archaea coexisting with diverse hosts in groundwater aquifers, and raise important questions about potential impacts on human health.

Cooperative protein folding requires distant regions of a protein to interact and provide mutual stabilization. The mechanism of this long-distance coupling remains poorly understood. Here, we use T4 lysozyme (T4L*) as a model to investigate long-range communications across two subdomains of a globular protein. T4L* is composed of two structurally distinct subdomains, although it behaves in a two-state manner at equilibrium. The subdomains of T4L* are connected via two topological connections: the N-terminal helix that is structurally part of the C-terminal subdomain (the A-helix) and a long helix that spans both subdomains (the C-helix). To understand the role that the C-helix plays in cooperative folding, we analyzed a circularly permuted version of T4L* (CP13*), whose subdomains are connected only by the C-helix. We demonstrate that when isolated as individual fragments, both subdomains of CP13* can fold autonomously into marginally stable conformations. The energetics of the N-terminal subdomain depend on the formation of a salt bridge known to be important for stability in the full-length protein. We show that the energetic contribution of the salt bridge to the stability of the N-terminal fragment increases when the C-helix is stabilized, such as occurs upon folding of the C-terminal subdomain. These results suggest a model where long-range energetic coupling is mediated by helix stabilization and not specific tertiary interactions.

Activation of T cells requires a global surge in cellular protein synthesis, accompanied by a large increase in translation initiation1–4. A central component of the translation initiation machinery–the multi-subunit eukaryotic initiation factor 3 (eIF3)–is rapidly turned on when quiescent T cells are stimulated3. However, the precise role eIF3 plays in activated T cells is not known. Using a global transcriptome crosslinking approach, we show human eIF3 interacts with a distinct set of mRNAs in activated Jurkat cells. A subset of these mRNAs, including those encoding the T cell receptor (TCR) subunits TCRA and TCRB, crosslink to eIF3 across the entire length of the mRNA. The TCRA and TCRB mRNAs do not co-localize with translationally repressed environments of P-bodies or stress granules but form distinct granules, potentially acting as translation “hot-spots.” T cell activation through CD28 causes a burst of TCR translation controlled by elements in the 3’-untranslated regions (3’-UTRs) of the TCRA and TCRB mRNAs that directly contact eIF3 and that are required for T cell activity. These results highlight a new role for eIF3 in regulating the translation dynamics of the TCR and provide insights that can guide the engineering of T cells used in cell immunotherapy applications.

The optimization of a new class of small molecule PCSK9 mRNA translation inhibitors is described. The potency, physicochemical properties, and off-target pharmacology associated with the hit compound (1) were improved by changes to two regions of the molecule. The last step in the synthesis of the congested amide center was enabled by three different routes. Subtle structural changes yielded significant changes in pharmacology and off-target margins. These efforts led to the identification of 7l and 7n with overall profiles suitable for in vivo evaluation. In a 14-day toxicology study, 7l demonstrated an improved safety profile vs lead 7f. We hypothesize that the improved safety profile is related to diminished binding of 7l to nontranslating ribosomes and an apparent improvement in transcript selectivity due to the lower strength of 7l stalling of off-target proteins.

Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single-molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we demonstrate that by combining purified and reconstituted in vitro translation, quantitative unnatural amino acid incorporation via AUG codon reassignment, and copper-catalyzed azide-alkyne cycloaddition, we can overcome these challenges for target proteins that are, or can be, methionine-depleted. We present an in vitro parallelizable approach that does not require laborious target-specific purification to generate dual-labeled proteins and ribosome-nascent chain libraries suitable for single-molecule FRET-based conformational phenotyping. We demonstrate the power of this approach by tracking the effects of mutations, C-terminal extensions, and ribosomal tethering on the structure and stability of three protein model systems: barnase, spectrin, and T4 lysozyme. Importantly, dual-labeled ribosome-nascent chain libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and should enable the in vitro directed evolution of proteins with designer single-molecule conformational phenotypes of interest.

By transplanting identity elements into E. coli tRNAfMet , we have engineered an orthogonal initiator tRNA (itRNATy2 ) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNATy2 can initiate translation in vivo with aromatic non-canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNAfMet from the genome afforded an E. coli strain in which the efficiency of non-canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.

Polypyrimidine tract-binding proteins (PTBPs) are RNA binding proteins that regulate a number of posttranscriptional events. Human PTBP1 transits between the nucleus and cytoplasm and is thought to regulate RNA processes in both. However, information about PTBP1 mRNA isoforms and regulation of PTPB1 expression remains incomplete. Here we mapped the major PTBP1 mRNA isoforms in HEK293T cells and identified alternative 5′ and 3′ untranslated regions (5′-UTRs, 3′-UTRs), as well as alternative splicing patterns in the protein coding region. We also assessed how the observed PTBP1 mRNA isoforms contribute to PTBP1 expression in different phases of the cell cycle. Previously, PTBP1 mRNAs were shown to crosslink to eukaryotic translation initiation factor 3 (eIF3). We find that eIF3 binds differently to each PTBP1 mRNA isoform in a cell cycle dependent manner. We also observe a strong correlation between eIF3 binding to PTBP1 mRNAs and repression of PTBP1 levels during the S phase of the cell cycle. Our results provide evidence of translational regulation of PTBP1 protein levels during the cell cycle, which may affect downstream regulation of alternative splicing and translation mediated by PTBP1 protein isoforms.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly expanding and largely untapped class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display an outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium sp. Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) – an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants belonging to the Rhizobiales. PHZ inhibits prokaryotic translation through the obstruction of the passage of the nascent peptide through the ribosome exit channel. The cryo-EM structure of the Escherichia coli ribosome with bound PHZ revealed that the drug interacts with the 23S rRNA and ribosomal proteins uL4 and uL22 and obstructs the exit tunnel in a way that is distinct from other compounds blocking the exit channel. We show that the sequence of uL4 ribosomal protein loop involved in PHZ binding determines the species-specificity of antibiotic interaction with its target. PHZ and its predicted homologs from other bacterial species expand the known diversity of LAPs and may be used in the future as biocontrol agents for the needs of agriculture.

Phage typically have small genomes and depend on their bacterial hosts for replication. DNA sequenced from many diverse ecosystems revealed hundreds of huge phage genomes, between 200 kbp and 716 kbp in length. Thirty-four genomes were manually curated to completion, including the largest phage genomes yet reported. Expanded genetic repertoires include diverse and new CRISPR-Cas systems, tRNAs, tRNA synthetases, tRNA modification enzymes, translation initiation and elongation factors, and ribosomal proteins. Phage CRISPR-Cas systems have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phage may repurpose bacterial CRISPR-Cas systems to eliminate competing phage. We phylogenetically define major clades of huge phage from human and other animal microbiomes, oceans, lakes, sediments, soils and the built environment. We conclude that their large gene inventories reflect a conserved biological strategy, observed over a broad bacterial host range and across Earth’s ecosystems.

Isomerases perform biotransformations without cofactors but often cause an undesirable mixture of substrate and product due to unfavorable thermodynamic equilibria. We demonstrate the feasibility of using an engineered yeast strain harboring oxidoreductase reactions to overcome the thermodynamic limit of an isomerization reaction. Specifically, a yeast strain capable of consuming lactose intracellularly is engineered to produce tagatose from lactose through three layers of manipulations. First, GAL1 coding for galactose kinase is deleted to eliminate galactose utilization. Second, heterologous xylose reductase (XR) and galactitol dehydrogenase (GDH) are introduced into the ∆gal1 strain. Third, the expression levels of XR and GDH are adjusted to maximize tagatose production. The resulting engineered yeast produces 37.69 g/L of tagatose from lactose with a tagatose and galactose ratio of 9:1 in the reaction broth. These results suggest that in vivo oxidoreaductase reactions can be employed to replace isomerases in vitro for biotransformation., A desired product cannot be obtained at higher concentration than its equilibrium concentration when isomerases are used for biotransformation. Here, the authors engineer in vivo oxidoreductive reactions in yeast to overcome the equilibrium limitation of in vitro isomerases-based tagatose production.

Ribosome-synthesized post-translationally modified peptides (RiPPs) represent a rapidly expanding class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) – an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants. The cryo-EM structure of the Escherichia coli 70S-PHZ complex reveals that the drug interacts with the 23S rRNA and uL4/uL22 proteins and obstructs ribosomal exit tunnel in a way that is distinct from other compounds. We show that the uL4 loop sequence determines the species-specificity of antibiotic action. PHZ expands the known diversity of LAPs and may be used in the future as biocontrol agent for agricultural needs., The authors report the identification of phazolicin (PHZ) - a prokaryotic translation inhibitory peptide - and its structure in complex with the E. coli ribosome, delineating PHZ’s mode of action and suggesting a basis for its bacterial species-specific activity.

When expressed in Saccharomyces cerevisiae using either of two constitutive yeast promoters ( PGK1 prom and CCW12 prom ), the transporters CDT-1 and CDT-2 from the filamentous fungus Neurospora crassa are able to catalyze, respectively, active transport and facilitated diffusion of cellobiose (and, for CDT-2, also xylan and its derivatives). In S. cerevisiae , endogenous permeases are removed from the plasma membrane by clathrin-mediated endocytosis and are marked for internalization through ubiquitinylation catalyzed by Rsp5, a HECT class ubiquitin:protein ligase (E3). Recruitment of Rsp5 to specific targets is mediated by a 14-member family of endocytic adaptor proteins, termed α-arrestins. Here we demonstrate that CDT-1 and CDT-2 are subject to α-arrestin-mediated endocytosis, that four α-arrestins (Rod1, Rog3, Aly1, and Aly2) are primarily responsible for this internalization, that the presence of the transport substrate promotes transporter endocytosis, and that, at least for CDT-2, residues located in its C-terminal cytosolic domain are necessary for its efficient endocytosis. Both α-arrestin-deficient cells expressing CDT-2 and otherwise wild-type cells expressing CDT-2 mutants unresponsive to α-arrestin-driven internalization exhibit an increased level of plasma membrane-localized transporter compared to that of wild-type cells, and they grow, utilize the transport substrate, and generate ethanol anaerobically better than control cells. IMPORTANCE Ethanolic fermentation of the breakdown products of plant biomass by budding yeast Saccharomyces cerevisiae remains an attractive biofuel source. To achieve this end, genes for heterologous sugar transporters and the requisite enzyme(s) for subsequent metabolism have been successfully expressed in this yeast. For one of the heterologous transporters examined in this study, we found that the amount of this protein residing in the plasma membrane was the rate-limiting factor for utilization of the cognate carbon source (cellobiose) and its conversion to ethanol.

Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although most prokaryotic adaptive immune systems generally target DNA substrates, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase). How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear. Here we show that bacterial C2c2 possesses a unique RNase activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated single-stranded RNA degradation activity. These dual RNase functions are chemically and mechanistically different from each other and from the crRNA-processing behaviour of the evolutionarily unrelated CRISPR enzyme Cpf1 (ref. 11). The two RNase activities of C2c2 enable multiplexed processing and loading of guide RNAs that in turn allow sensitive detection of cellular transcripts.

Eukaryotic mRNAs contain a 5′ cap structure that is crucial for recruitment of the translation machinery and initiation of protein synthesis. mRNA recognition is thought to require direct interactions between eukaryotic initiation factor 4E (eIF4E) and the mRNA cap. However, translation of numerous capped mRNAs remains robust during cellular stress, early development, and cell cycle progression despite inactivation of eIF4E. Here we describe a cap-dependent pathway of translation initiation in human cells that relies on a previously unknown cap-binding activity of eIF3d, a subunit of the 800-kilodalton eIF3 complex. A 1.4 Å crystal structure of the eIF3d cap-binding domain reveals unexpected homology to endonucleases involved in RNA turnover, and allows modelling of cap recognition by eIF3d. eIF3d makes specific contacts with the cap, as exemplified by cap analogue competition, and these interactions are essential for assembly of translation initiation complexes on eIF3-specialized mRNAs such as the cell proliferation regulator c-Jun (also known as JUN). The c-Jun mRNA further encodes an inhibitory RNA element that blocks eIF4E recruitment, thus enforcing alternative cap recognition by eIF3d. Our results reveal a mechanism of cap-dependent translation that is independent of eIF4E, and illustrate how modular RNA elements work together to direct specialized forms of translation initiation.

The production of bioenergy from plant biomass previously relied on using microorganisms that rapidly and efficiently convert simple sugars into fuels and chemicals. However, to exploit the far more abundant carbon fixed in plant cell walls, future industrial production hosts will need to be engineered to leverage the most efficient biochemical pathways and most robust traits that can be found in nature. The CRISPR-Cas9 genome editing technology now enables writing the genome at will, which will allow biotechnology to become an 'information science.' This review covers recent advances in using CRISPR-Cas9 to engineer the genomes of a wide variety of organisms that could be use in the industrial production of biofuels and renewable chemicals.

Economical biofuel production from plant biomass requires the conversion of both cellulose and hemicellulose in the plant cell wall. The best industrial fermentation organism, the yeast Saccharomyces cerevisiae, has been developed to utilize xylose by heterologously expressing either a xylose reductase/xylitol dehydrogenase (XR/XDH) pathway or a xylose isomerase (XI) pathway. Although it has been proposed that the optimal means for fermenting xylose into biofuels would use XI instead of the XR/XDH pathway, no clear comparison of the best publicly-available yeast strains engineered to use XR/XDH or XI has been published. We therefore compared two of the best-performing engineered yeast strains in the public domain—one using the XR/XDH pathway and another using XI—in anaerobic xylose fermentations. We find that, regardless of conditions, the strain using XR/XDH has substantially higher productivity compared to the XI strain. By contrast, the XI strain has better yields in nearly all conditions tested.

IRES-mediated translation of key cell fate regulating genes has been implicated in tumorigenesis. Concerted action of canonical eukaryotic initiation factors and IRES transacting factors (ITAFs) was shown to regulate cellular IRES mediated translation; however, the precise molecular mechanism of ribosome recruitment to cellular IRESes remains unclear. Here we show that the X-linked inhibitor of apoptosis (XIAP) IRES operates in an evolutionary conserved viral like mode and the structural integrity, particularly in the vicinity of AUG, is critical for ribosome recruitment. The binding of eIF3 together with PABP potentiates ribosome recruitment to the IRES. Our data support the model in which eIF3 binds directly to the XIAP IRES RNA in a structure-dependent manner and acts as a scaffold for IRES RNA, PABP and the 40S ribosome.

© 2016 Biophysical Society. Poor consistency of the ice thickness from one area of a cryo-electron microscope (cryo-EM) specimen grid to another, from one grid to the next, and from one type of specimen to another, motivates a reconsideration of how to best prepare suitably thin specimens. Here we first review the three related topics of wetting, thinning, and stability against dewetting of aqueous films spread over a hydrophilic substrate. We then suggest that the importance of there being a surfactant monolayer at the air-water interface of thin, cryo-EM specimens has been largely underappreciated. In fact, a surfactant layer (of uncontrolled composition and surface pressure) can hardly be avoided during standard cryo-EM specimen preparation. We thus suggest that better control over the composition and properties of the surfactant layer may result in more reliable production of cryo-EM specimens with the desired thickness.