Structural basis for human mitochondrial tRNA maturation.

The human mitochondrial genome is transcribed into two RNAs, containing mRNAs, rRNAs and tRNAs, all dedicated to produce essential proteins of the respiratory chain. The precise excision of tRNAs by the mitochondrial endoribonucleases (mt-RNase), P and Z, releases all RNA species from the two RNA transcripts. The tRNAs then undergo 3'-CCA addition. In metazoan mitochondria, RNase P is a multi-enzyme assembly that comprises the endoribonuclease PRORP and a tRNA methyltransferase subcomplex. The requirement for this tRNA methyltransferase subcomplex for mt-RNase P cleavage activity, as well as the mechanisms of pre-tRNA 3'-cleavage and 3'-CCA addition, are still poorly understood. Here, we report cryo-EM structures that visualise four steps of mitochondrial tRNA maturation: 5' and 3' tRNA-end processing, methylation and 3'-CCA addition, and explain the defined sequential order of the tRNA processing steps. The methyltransferase subcomplex recognises the pre-tRNA in a distinct mode that can support tRNA-end processing and 3'-CCA addition, likely resulting from an evolutionary adaptation of mitochondrial tRNA maturation complexes to the structurally-fragile mitochondrial tRNAs. This subcomplex can also ensure a tRNA-folding quality-control checkpoint before the sequential docking of the maturation enzymes. Altogether, our study provides detailed molecular insight into RNA-transcript processing and tRNA maturation in human mitochondria.

Cooperation of regulatory RNA and the RNA degradosome in transcript surveillance.

The ompD transcript, encoding an outer membrane porin in Salmonella, harbors a controlling element in its coding region that base-pairs imperfectly with a 'seed' region of the small regulatory RNA (sRNA) MicC. When tagged with the sRNA, the ompD mRNA is cleaved downstream of the pairing site by the conserved endoribonuclease RNase E, leading to transcript destruction. We observe that the sRNA-induced cleavage site is accessible to RNase E in vitro upon recruitment of ompD into the 30S translation pre-initiation complex (PIC) in the presence of the degradosome components. Evaluation of substrate accessibility suggests that the paused 30S PIC presents the mRNA for targeted recognition and degradation. Ribonuclease activity on PIC-bound ompD is critically dependent on the recruitment of RNase E into the multi-enzyme RNA degradosome, and our data suggest a process of substrate capture and handover to catalytic sites within the degradosome, in which sequential steps of seed matching and duplex remodelling contribute to cleavage efficiency. Our findings support a putative mechanism of surveillance at translation that potentially terminates gene expression efficiently and rapidly in response to signals provided by regulatory RNA.

Acetylation regulates the oligomerization state and activity of RNase J, the Helicobacter pylori major ribonuclease.

In the gastric pathogen Helicobacter pylori, post-transcriptional regulation relies strongly on the activity of the essential ribonuclease RNase J. Here, we elucidated the crystal and cryo-EM structures of RNase J and determined that it assembles into dimers and tetramers in vitro. We found that RNase J extracted from H. pylori is acetylated on multiple lysine residues. Alanine substitution of several of these residues impacts on H. pylori morphology, and thus on RNase J function in vivo. Mutations of Lysine 649 modulates RNase J oligomerization in vitro, which in turn influences ribonuclease activity in vitro. Our structural analyses of RNase J reveal loops that gate access to the active site and rationalizes how acetylation state of K649 can influence activity. We propose acetylation as a regulatory level controlling the activity of RNase J and its potential cooperation with other enzymes of RNA metabolism in H. pylori.

Catabolite repression control protein antagonist, a novel player in Pseudomonas aeruginosa carbon catabolite repression control.

In the opportunistic human pathogen Pseudomonas aeruginosa (Pae), carbon catabolite repression (CCR) orchestrates the hierarchical utilization of N and C sources, and impacts virulence, antibiotic resistance and biofilm development. During CCR, the RNA chaperone Hfq and the catabolite repression control protein Crc form assemblies on target mRNAs that impede translation of proteins involved in uptake and catabolism of less preferred C sources. After exhaustion of the preferred C-source, translational repression of target genes is relieved by the regulatory RNA CrcZ, which binds to and acts as a decoy for Hfq. Here, we asked whether Crc action can be modulated to relieve CCR after exhaustion of a preferred carbon source. As Crc does not bind to RNA per se, we endeavored to identify an interacting protein. In vivo co-purification studies, co-immunoprecipitation and biophysical assays revealed that Crc binds to Pae strain O1 protein PA1677. Our structural studies support bioinformatics analyzes showing that PA1677 belongs to the isochorismatase-like superfamily. Ectopic expression of PA1677 resulted in de-repression of Hfq/Crc controlled target genes, while in the absence of the protein, an extended lag phase is observed during diauxic growth on a preferred and a non-preferred carbon source. This observations indicate that PA1677 acts as an antagonist of Crc that favors synthesis of proteins required to metabolize non-preferred carbon sources. We present a working model wherein PA1677 diminishes the formation of productive Hfq/Crc repressive complexes on target mRNAs by titrating Crc. Accordingly, we propose the name CrcA (catabolite repression control protein antagonist) for PA1677.

Structure of a bacterial ribonucleoprotein complex central to the control of cell envelope biogenesis.

Biogenesis of the essential precursor of the bacterial cell envelope, glucosamine-6-phosphate (GlcN6P), is controlled by intricate post-transcriptional networks mediated by GlmZ, a small regulatory RNA (sRNA). GlmZ stimulates translation of the mRNA encoding GlcN6P synthtase in Escherichia coli, but when bound by RapZ protein, the sRNA becomes inactivated through cleavage by the endoribonuclease RNase E. Here, we report the cryoEM structure of the RapZ:GlmZ complex, revealing a complementary match of the RapZ tetrameric quaternary structure to structural repeats in the sRNA. The nucleic acid is contacted by RapZ mostly through a highly conserved domain that shares an evolutionary relationship with phosphofructokinase and suggests links between metabolism and riboregulation. We also present the structure of a precleavage intermediate formed between the binary RapZ:GlmZ complex and RNase E that reveals how GlmZ is presented and recognised by the enzyme. The structures provide a framework for understanding how other encounter complexes might guide recognition and action of endoribonucleases on target transcripts, and how structured substrates in polycistronic precursors may be recognised for processing by RNase E.

Translational regulation by Hfq-Crc assemblies emerges from polymorphic ribonucleoprotein folding.

The widely occurring bacterial RNA chaperone Hfq is a key factor in the post-transcriptional control of hundreds of genes in Pseudomonas aeruginosa. How this broadly acting protein can contribute to the regulatory requirements of many different genes remains puzzling. Here, we describe cryo-EM structures of higher order assemblies formed by Hfq and its partner protein Crc on control regions of different P. aeruginosa target mRNAs. Our results show that these assemblies have mRNA-specific quaternary architectures resulting from the combination of multivalent protein-protein interfaces and recognition of patterns in the RNA sequence. The structural polymorphism of these ribonucleoprotein assemblies enables selective translational repression of many different target mRNAs. This system elucidates how highly complex regulatory pathways can evolve with a minimal economy of proteinogenic components in combination with RNA sequence and fold.

Acetylation of a fungal effector that translocates host PR1 facilitates virulence.

Pathogens utilize a panoply of effectors to manipulate plant defense. However, despite their importance, relatively little is actually known about regulation of these virulence factors. Here, we show that the effector Fol-Secreted Virulence-related Protein1 (FolSvp1), secreted from fungal pathogen Fusarium oxysporum f. sp. lycopersici (Fol), directly binds and translocates the tomato pathogenesis-related protein1, SlPR1, from the apoplast outside the plasma membrane to the host nucleus via its nuclear localization signal. Relocation of SlPR1 abolishes generation of the defense signaling peptide, CAPE1, from its C-terminus, and as a consequence, facilitates pathogen invasion of plants. The action of FolSvp1 requires covalent modification by acetylation for full virulence in host tomato tissues. The modification is catalyzed by the Fol FolArd1 lysine acetyltransferase prior to secretion. Addition of an acetyl group to one residue, K167, prevents ubiquitination-dependent degradation of FolSvp1 in both Fol and plant cells with different mechanisms, allowing it to function normally in fungal invasion. Either inactivation of FolSvp1 or removal of the acetyl group on K167 leads to impaired pathogenicity of Fol. These findings indicate that acetylation can regulate the stability of effectors of fungal plant pathogens with impact on virulence.

Broad-spectrum chemicals block ROS detoxification to prevent plant fungal invasion.

Plant diseases cause a huge impact on food security and are of global concern. While application of agrochemicals is a common approach in the control of plant diseases currently, growing drug resistance and the impact of off-target effects of these compounds pose major challenges. The identification of pathogenicity-related virulence mechanisms and development of new chemicals that target these processes are urgently needed. One such virulence mechanism is the detoxification of reactive oxygen species (ROS) generated by host plants upon attack by pathogens. The machinery of ROS detoxification might therefore serve as a drug target for preventing plant diseases, but few anti-ROS-scavenging drugs have been developed. Here, we show that in the model system Botrytis cinerea secretion of the cytochrome c-peroxidase, BcCcp1 removes plant-produced H2O2 and promotes pathogen invasion. The peroxidase secretion is modulated by a Tom1-like protein, BcTol1, through physical interaction. We show that BcTol1 is regulated at different levels to enhance the secretion of BcCcp1 during the early infection stage. Inactivation of either BcTol1 or BcCcp1 leads to dramatically reduced virulence of B. cinerea. We identify two BcTol1-targeting small molecules that not only prevent B. cinerea invasion but also have effective activity against a wide range of plant fungal pathogens without detectable effect on the hosts. These findings reveal a conserved mechanism of ROS detoxification in fungi and provide a class of potential fungicides to control diverse plant diseases. The approach described here has wide implications for further drug discovery in related fields.

Molecular biology for green recovery-A call for action.

Molecular biology holds a vast potential for tackling climate change and biodiversity loss. Yet, it is largely absent from the current strategies. We call for a community-wide action to bring molecular biology to the forefront of climate change solutions.

Flipping and other astonishing transporter dance moves in fungal drug resistance.

In all domains of life, transmembrane proteins from the ATP-binding cassette (ABC) transporter family drive the translocation of diverse substances across lipid bilayers. In pathogenic fungi, the ABC transporters of the pleiotropic drug resistance (PDR) subfamily confer antibiotic resistance and so are of interest as therapeutic targets. They also drive the quest for understanding how ABC transporters can generally accommodate such a wide range of substrates. The Pdr5 transporter from baker's yeast is representative of the PDR group and, ever since its discovery more than 30 years ago, has been the subject of extensive functional analyses. A new perspective of these studies has been recently provided in the framework of the first electron cryo-microscopy structures of Pdr5, as well as emergent applications of machine learning in the field. Taken together, the old and the new developments have been used to propose a mechanism for the transport process in PDR proteins. This mechanism involves a "flippase" step that moves the substrates from one leaflet of the bilayer to the other, as a central element of cellular efflux.

Cryo-EM structures of staphylococcal IsdB bound to human hemoglobin reveal the process of heme extraction.

Iron surface determinant B (IsdB) is a hemoglobin (Hb) receptor essential for hemic iron acquisition by Staphylococcus aureus. Heme transfer to IsdB is possible from oxidized Hb (metHb), but inefficient from Hb either bound to oxygen (oxyHb) or bound to carbon monoxide (HbCO), and encompasses a sequence of structural events that are currently poorly understood. By single-particle cryo-electron microscopy, we determined the structure of two IsdB:Hb complexes, representing key species along the heme extraction pathway. The IsdB:HbCO structure, at 2.9-Å resolution, provides a snapshot of the preextraction complex. In this early stage of IsdB:Hb interaction, the hemophore binds to the ß-subunits of the Hb tetramer, exploiting a folding-upon-binding mechanism that is likely triggered by a cis/trans isomerization of Pro173. Binding of IsdB to α-subunits occurs upon dissociation of the Hb tetramer into α/ß dimers. The structure of the IsdB:metHb complex reveals the final step of the extraction process, where heme transfer to IsdB is completed. The stability of the complex, both before and after heme transfer from Hb to IsdB, is influenced by isomerization of Pro173. These results greatly enhance current understanding of structural and dynamic aspects of the heme extraction mechanism by IsdB and provide insight into the interactions that stabilize the complex before the heme transfer event. This information will support future efforts to identify inhibitors of heme acquisition by S. aureus by interfering with IsdB:Hb complex formation.

A Model for Allosteric Communication in Drug Transport by the AcrAB-TolC Tripartite Efflux Pump.

RND family efflux pumps are complex macromolecular machines involved in multidrug resistance by extruding antibiotics from the cell. While structural studies and molecular dynamics simulations have provided insights into the architecture and conformational states of the pumps, the path followed by conformational changes from the inner membrane protein (IMP) to the periplasmic membrane fusion protein (MFP) and to the outer membrane protein (OMP) in tripartite efflux assemblies is not fully understood. Here, we investigated AcrAB-TolC efflux pump's allostery by comparing resting and transport states using difference distance matrices supplemented with evolutionary couplings data and buried surface area measurements. Our analysis indicated that substrate binding by the IMP triggers quaternary level conformational changes in the MFP, which induce OMP to switch from the closed state to the open state, accompanied by a considerable increase in the interface area between the MFP subunits and between the OMPs and MFPs. This suggests that the pump's transport-ready state is at a more favourable energy level than the resting state, but raises the puzzle of how the pump does not become stably trapped in a transport-intermediate state. We propose a model for pump allostery that includes a downhill energetic transition process from a proposed 'activated' transport state back to the resting pump.

Multi-scale ensemble properties of the Escherichia coli RNA degradosome.

In organisms from all domains of life, multi-enzyme assemblies play central roles in defining transcript lifetimes and facilitating RNA-mediated regulation of gene expression. An assembly dedicated to such roles, known as the RNA degradosome, is found amongst bacteria from highly diverse lineages. About a fifth of the assembly mass of the degradosome of Escherichia coli and related species is predicted to be intrinsically disordered - a property that has been sustained for over a billion years of bacterial molecular history and stands in marked contrast to the high degree of sequence variation of that same region. Here, we characterize the conformational dynamics of the degradosome using a hybrid structural biology approach that combines solution scattering with ad hoc ensemble modelling, cryo-electron microscopy, and other biophysical methods. The E. coli degradosome can form punctate bodies in vivo that may facilitate its functional activities, and based on our results, we propose an electrostatic switch model to account for the propensity of the degradosome to undergo programmable puncta formation.

In situ structure of the AcrAB-TolC efflux pump at subnanometer resolution.

The tripartite AcrAB-TolC assembly, which spans both the inner and outer membranes in Gram-negative bacteria, is an efflux pump that contributes to multidrug resistance. Here, we present the in situ structure of full-length Escherichia coli AcrAB-TolC determined at 7 Å resolution by electron cryo-tomography. The TolC channel penetrates the outer membrane bilayer through to the outer leaflet and exhibits two different configurations that differ by a 60° rotation relative to the AcrB position in the pump assembly. AcrA protomers interact directly with the inner membrane and with AcrB via an interface located in proximity to the AcrB ligand-binding pocket. Our structural analysis suggests that these AcrA-bridged interactions underlie an allosteric mechanism for transmitting drug-evoked signals from AcrB to the TolC channel within the pump. Our study demonstrates the power of in situ electron cryo-tomography, which permits critical insights into the function of bacterial efflux pumps.

The decrotonylase FoSir5 facilitates mitochondrial metabolic state switching in conidial germination of Fusarium oxysporum.

Fusarium oxysporum is one of the most important pathogenic fungi with a broad range of plant and animal hosts. The first key step of its infection cycle is conidial germination, but there is limited information available on the molecular events supporting this process. We show here that germination is accompanied by a sharp decrease in expression of FoSir5, an ortholog of the human lysine deacetylase SIRT5. We observe that FoSir5 decrotonylates a subunit of the fungal pyruvate dehydrogenase complex (FoDLAT) at K148, resulting in inhibition of the activity of the complex in mitochondria. Moreover, FoSir5 decrotonylates histone H3K18, leading to a downregulation of transcripts encoding enzymes of aerobic respiration pathways. Thus, the activity of FoSir5 coordinates regulation in different organelles to steer metabolic flux through respiration. As ATP content is positively related to fungal germination, we propose that FoSir5 negatively modulates conidial germination in F. oxysporum through its metabolic impact. These findings provide insights into the multifaceted roles of decrotonylation, catalyzed by FoSir5, that support conidial germination in F. oxysporum.

Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5.

Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Pdr5 and its homologues drive drug efflux through uncoupled hydrolysis of nucleotides, enabling organisms such as baker's yeast and pathogenic fungi to survive in the presence of chemically diverse antifungal agents. Here, we present the molecular structure of Pdr5 solved with single particle cryo-EM, revealing details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across inter-domain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens new avenues for the development of effective antifungal compounds.

Impact of pseudouridylation, substrate fold, and degradosome organization on the endonuclease activity of RNase E.

The conserved endoribonuclease RNase E dominates the dynamic landscape of RNA metabolism and underpins control mediated by small regulatory RNAs in diverse bacterial species. We explored the enzyme's hydrolytic mechanism, allosteric activation, and interplay with partner proteins in the multicomponent RNA degradosome assembly of Escherichia coli. RNase E cleaves single-stranded RNA with preference to attack the phosphate located at the 5' nucleotide preceding uracil, and we corroborate key interactions that select that base. Unexpectedly, RNase E activity is impeded strongly when the recognized uracil is isomerized to 5-ribosyluracil (pseudouridine), from which we infer the detailed geometry of the hydrolytic attack process. Kinetics analyses support models for recognition of secondary structure in substrates by RNase E and for allosteric autoregulation. The catalytic power of the enzyme is boosted when it is assembled into the multienzyme RNA degradosome, most likely as a consequence of substrate capture and presentation. Our results rationalize the origins of substrate preferences of RNase E and illuminate its catalytic mechanism, supporting the roles of allosteric domain closure and cooperation with other components of the RNA degradosome complex.

A cooperative PNPase-Hfq-RNA carrier complex facilitates bacterial riboregulation.

Polynucleotide phosphorylase (PNPase) is an ancient exoribonuclease conserved in the course of evolution and is found in species as diverse as bacteria and humans. Paradoxically, Escherichia coli PNPase can act not only as an RNA degrading enzyme but also by an unknown mechanism as a chaperone for small regulatory RNAs (sRNAs), with pleiotropic consequences for gene regulation. We present structures of the ternary assembly formed by PNPase, the RNA chaperone Hfq, and sRNA and show that this complex boosts sRNA stability in vitro. Comparison of structures for PNPase in RNA carrier and degradation modes reveals how the RNA is rerouted away from the active site through interactions with Hfq and the KH and S1 domains. Together, these data explain how PNPase is repurposed to protect sRNAs from cellular ribonucleases such as RNase E and could aid RNA presentation to facilitate regulatory actions on target genes.

Targeting the Conserved Stem Loop 2 Motif in the SARS-CoV-2 Genome.

RNA structural elements occur in numerous single-stranded positive-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3' untranslated region (UTR) in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts imply a key role for s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodeled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 replication in cell culture. Our results thus permit us to suggest that the s2m element is readily targeted by ASOs, which show promise as antiviral agents. IMPORTANCE The highly conserved stem-loop 2 motif (s2m) is found in the genomes of many RNA viruses, including SARS-CoV-2. Our findings indicate that the s2m element can be targeted by antisense oligonucleotides. The antiviral potential of this element represents a promising start for further research into targeting conserved elements in RNA viruses.

MD simulations reveal the basis for dynamic assembly of Hfq-RNA complexes.

The conserved protein Hfq is a key factor in the RNA-mediated control of gene expression in most known bacteria. The transient intermediates Hfq forms with RNA support intricate and robust regulatory networks. In Pseudomonas, Hfq recognizes repeats of adenine-purine-any nucleotide (ARN) in target mRNAs via its distal binding side, and together with the catabolite repression control (Crc) protein, assembles into a translation-repression complex. Earlier experiments yielded static, ensemble-averaged structures of the complex, but details of its interface dynamics and assembly pathway remained elusive. Using explicit solvent atomistic molecular dynamics simulations, we modeled the extensive dynamics of the Hfq-RNA interface and found implications for the assembly of the complex. We predict that syn/anti flips of the adenine nucleotides in each ARN repeat contribute to a dynamic recognition mechanism between the Hfq distal side and mRNA targets. We identify a previously unknown binding pocket that can accept any nucleotide and propose that it may serve as a 'status quo' staging point, providing nonspecific binding affinity, until Crc engages the Hfq-RNA binary complex. The dynamical components of the Hfq-RNA recognition can speed up screening of the pool of the surrounding RNAs, participate in rapid accommodation of the RNA on the protein surface, and facilitate competition among different RNAs. The register of Crc in the ternary assembly could be defined by the recognition of a guanine-specific base-phosphate interaction between the first and last ARN repeats of the bound RNA. This dynamic substrate recognition provides structural rationale for the stepwise assembly of multicomponent ribonucleoprotein complexes nucleated by Hfq-RNA binding.