Publications by authors named "Ben F Luisi"

107 Publications

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

Mol Cell 2021 07 21;81(14):2901-2913.e5. Epub 2021 Jun 21.

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molcel.2021.05.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8294330PMC
July 2021

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

J Virol 2021 06 24;95(14):e0066321. Epub 2021 Jun 24.

Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.

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. 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.
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http://dx.doi.org/10.1128/JVI.00663-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8223950PMC
June 2021

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

J Biol Chem 2021 Jan-Jun;296:100656. Epub 2021 Apr 20.

Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic.

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.
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http://dx.doi.org/10.1016/j.jbc.2021.100656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8121710PMC
April 2021

Protein Pulldown Assays to Monitor the Composition of the Bacterial RNA Degradosome.

Methods Mol Biol 2021 ;2209:425-432

Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil.

The method of co-immunoprecipitation (co-IP or pulldown) enables the identification of proteins interacting in macromolecular assemblies, through the purification of a key protein by affinity chromatography using specific antibodies immobilized on a matrix. The advantages of using epitope-tagged proteins include the ability to use commercially available antibodies for affinity purifications, and typically they do not disrupt the structure of the protein complexes. Here we describe the utilization of an epitope-tagged version of Caulobacter crescentus RNase E in order to determine the composition of the RNA degradosome under different growth conditions. Several proteins that interact with the RNA degradosome were identified.
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http://dx.doi.org/10.1007/978-1-0716-0935-4_26DOI Listing
March 2021

Structures of B. subtilis Maturation RNases Captured on 50S Ribosome with Pre-rRNAs.

Mol Cell 2020 10 28;80(2):227-236.e5. Epub 2020 Sep 28.

Expression Génétique Microbienne, UMR 8261, CNRS, Université de Paris, Institut de Biologie Physico-Chimique (IBPC), 75005 Paris, France. Electronic address:

The pathways for ribosomal RNA (rRNA) maturation diverge greatly among the domains of life. In the Gram-positive model bacterium, Bacillus subtilis, the final maturation steps of the two large ribosomal subunit (50S) rRNAs, 23S and 5S pre-rRNAs, are catalyzed by the double-strand specific ribonucleases (RNases) Mini-RNase III and RNase M5, respectively. Here we present a protocol that allowed us to solve the 3.0 and 3.1 Å resolution cryoelectron microscopy structures of these RNases poised to cleave their pre-rRNA substrates within the B. subtilis 50S particle. These data provide the first structural insights into rRNA maturation in bacteria by revealing how these RNases recognize and process double-stranded pre-rRNA. Our structures further uncover how specific ribosomal proteins act as chaperones to correctly fold the pre-rRNA substrates and, for Mini-III, anchor the RNase to the ribosome. These r-proteins thereby serve a quality-control function in the process from accurate ribosome assembly to rRNA processing.
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http://dx.doi.org/10.1016/j.molcel.2020.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610893PMC
October 2020

Computational design of transmembrane pores.

Nature 2020 09 26;585(7823):129-134. Epub 2020 Aug 26.

Institute for Protein Design, University of Washington, Seattle, WA, USA.

Transmembrane channels and pores have key roles in fundamental biological processes and in biotechnological applications such as DNA nanopore sequencing, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels, and there have been recent advances in de novo membrane protein design and in redesigning naturally occurring channel-containing proteins. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.
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http://dx.doi.org/10.1038/s41586-020-2646-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483984PMC
September 2020

Interactions of a Bacterial RND Transporter with a Transmembrane Small Protein in a Lipid Environment.

Structure 2020 06 28;28(6):625-634.e6. Epub 2020 Apr 28.

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK. Electronic address:

The small protein AcrZ in Escherichia coli interacts with the transmembrane portion of the multidrug efflux pump AcrB and increases resistance of the bacterium to a subset of the antibiotic substrates of that transporter. It is not clear how the physical association of the two proteins selectively changes activity of the pump for defined substrates. Here, we report cryo-EM structures of AcrB and the AcrBZ complex in lipid environments, and comparisons suggest that conformational changes occur in the drug-binding pocket as a result of AcrZ binding. Simulations indicate that cardiolipin preferentially interacts with the AcrBZ complex, due to increased contact surface, and we observe that chloramphenicol sensitivity of bacteria lacking AcrZ is exacerbated when combined with cardiolipin deficiency. Taken together, the data suggest that AcrZ and lipid cooperate to allosterically modulate AcrB activity. This mode of regulation by a small protein and lipid may occur for other membrane proteins.
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http://dx.doi.org/10.1016/j.str.2020.03.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7267776PMC
June 2020

RNA lifetime control, from stereochemistry to gene expression.

Curr Opin Struct Biol 2020 04 21;61:59-70. Epub 2019 Dec 21.

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK. Electronic address:

Through the activities of various multi-component assemblies, protein-coding transcripts can be chaperoned toward protein synthesis or nudged into a funnel of rapid destruction. The capacity of these machine-like assemblies to tune RNA lifetime underpins the harmony of gene expression in all cells. Some of the molecular machines that mediate transcript turnover also contribute to on-the-fly surveillance of aberrant mRNAs and non-coding RNAs. How these dynamic assemblies distinguish functional RNAs from those that must be degraded is an intriguing puzzle for understanding the regulation of gene expression and dysfunction associated with disease. Recent data illuminate what the machines look like, and how they find, recognise and operate on transcripts to sculpt the dynamic regulatory landscape. This review captures current structural and mechanistic insights into the key enzymes and their effector assemblies that contribute to the fate-determining decision points for RNA in post-transcriptional control of genetic information.
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http://dx.doi.org/10.1016/j.sbi.2019.10.002DOI Listing
April 2020

In situ structure and assembly of the multidrug efflux pump AcrAB-TolC.

Nat Commun 2019 06 14;10(1):2635. Epub 2019 Jun 14.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, 77030, USA.

Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors.
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http://dx.doi.org/10.1038/s41467-019-10512-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570770PMC
June 2019

Hfq structure reveals a conserved mechanism of RNA annealing regulation.

Proc Natl Acad Sci U S A 2019 05 10;116(22):10978-10987. Epub 2019 May 10.

Department of Biochemistry, University of Cambridge, CB2 1GA Cambridge, United Kingdom

We have solved the X-ray crystal structure of the RNA chaperone protein Hfq from the alpha-proteobacterium to 2.15-Å resolution, resolving the conserved core of the protein and the entire C-terminal domain (CTD). The structure reveals that the CTD of neighboring hexamers pack in crystal contacts, and that the acidic residues at the C-terminal tip of the protein interact with positive residues on the rim of Hfq, as has been recently proposed for a mechanism of modulating RNA binding. De novo computational models predict a similar docking of the acidic tip residues against the core of Hfq. We also show that Hfq has sRNA binding and RNA annealing activities and is capable of facilitating the annealing of certain sRNA:mRNA pairs in vivo. Finally, we describe how the Hfq CTD and its acidic tip residues provide a mechanism to modulate annealing activity and substrate specificity in various bacteria.
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http://dx.doi.org/10.1073/pnas.1814428116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561178PMC
May 2019

Control of Bacterial Virulence through the Peptide Signature of the Habitat.

Cell Rep 2019 02;26(7):1815-1827.e5

Microbial Pathogenesis Group, Infection Medicine, Edinburgh Medical School (Biomedical Sciences) and The Roslin Institute, University of Edinburgh, Edinburgh EH16 4SB, UK. Electronic address:

To optimize fitness, pathogens selectively activate their virulence program upon host entry. Here, we report that the facultative intracellular bacterium Listeria monocytogenes exploits exogenous oligopeptides, a ubiquitous organic N source, to sense the environment and control the activity of its virulence transcriptional activator, PrfA. Using a genetic screen in adsorbent-treated (PrfA-inducing) medium, we found that PrfA is functionally regulated by the balance between activating and inhibitory nutritional peptides scavenged via the Opp transport system. Activating peptides provide essential cysteine precursor for the PrfA-inducing cofactor glutathione (GSH). Non-cysteine-containing peptides cause promiscuous PrfA inhibition. Biophysical and co-crystallization studies reveal that peptides inhibit PrfA through steric blockade of the GSH binding site, a regulation mechanism directly linking bacterial virulence and metabolism. L. monocytogenes mutant analysis in macrophages and our functional data support a model in which changes in the balance of antagonistic Opp-imported oligopeptides promote PrfA induction intracellularly and PrfA repression outside the host.
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http://dx.doi.org/10.1016/j.celrep.2019.01.073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389498PMC
February 2019

Architectural principles for Hfq/Crc-mediated regulation of gene expression.

Elife 2019 02 13;8. Epub 2019 Feb 13.

Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.

In diverse bacterial species, the global regulator Hfq contributes to post-transcriptional networks that control expression of numerous genes. Hfq of the opportunistic pathogen inhibits translation of target transcripts by forming a regulatory complex with the catabolite repression protein Crc. This repressive complex acts as part of an intricate mechanism of preferred nutrient utilisation. We describe high-resolution cryo-EM structures of the assembly of Hfq and Crc bound to the translation initiation site of a target mRNA. The core of the assembly is formed through interactions of two cognate RNAs, two Hfq hexamers and a Crc pair. Additional Crc protomers are recruited to the core to generate higher-order assemblies with demonstrated regulatory activity in vivo. This study reveals how Hfq cooperates with a partner protein to regulate translation, and provides a structural basis for an RNA code that guides global regulators to interact cooperatively and regulate different RNA targets.
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http://dx.doi.org/10.7554/eLife.43158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6422490PMC
February 2019

Substrate Recognition and Autoinhibition in the Central Ribonuclease RNase E.

Mol Cell 2018 10 27;72(2):275-285.e4. Epub 2018 Sep 27.

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK. Electronic address:

The endoribonuclease RNase E is a principal factor in RNA turnover and processing that helps to exercise fine control of gene expression in bacteria. While its catalytic activity can be strongly influenced by the chemical identity of the 5' end of RNA substrates, the enzyme can also cleave numerous substrates irrespective of the chemistry of their 5' ends through a mechanism that has remained largely unexplained. We report structural and functional data illuminating details of both operational modes. Our crystal structure of RNase E in complex with the sRNA RprA reveals a duplex recognition site that saddles an inter-protomer surface to help present substrates for cleavage. Our data also reveal an autoinhibitory pocket that modulates the overall activity of the ribonuclease. Taking these findings together, we propose how RNase E uses versatile modes of RNA recognition to achieve optimal activity and specificity.
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http://dx.doi.org/10.1016/j.molcel.2018.08.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202311PMC
October 2018

Author Correction: Multidrug efflux pumps: structure, function and regulation.

Nat Rev Microbiol 2018 Sep;16(9):577

Department of Biochemistry, University of Cambridge, Cambridge, UK.

In the version of this Review originally published, the author contributions of co-author Arthur Neuberger were incorrectly listed. The author contributions should have appeared as 'D.D., X.W.-K., A.N., H.W.v.V., K.M.P., L.J.V.P. and B.F.L. researched data for the article, made substantial contributions to discussions of the content, wrote the article, and reviewed and edited the manuscript before submission'. This has now been corrected in all versions of the Review. The authors apologize to readers for this error.
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http://dx.doi.org/10.1038/s41579-018-0060-xDOI Listing
September 2018

Multidrug efflux pumps: structure, function and regulation.

Nat Rev Microbiol 2018 09;16(9):523-539

Department of Biochemistry, University of Cambridge, Cambridge, UK.

Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this Review, we describe recent advances that have increased our understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria. Clinical and laboratory data indicate that efflux pumps function not only in the drug extrusion process but also in virulence and the adaptive responses that contribute to antimicrobial resistance during infection. The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities.
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http://dx.doi.org/10.1038/s41579-018-0048-6DOI Listing
September 2018

Structure and mechanism of bacterial tripartite efflux pumps.

Res Microbiol 2018 Sep - Oct;169(7-8):401-413. Epub 2018 May 19.

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK. Electronic address:

Efflux pumps are membrane proteins which contribute to multi-drug resistance. In Gram-negative bacteria, some of these pumps form complex tripartite assemblies in association with an outer membrane channel and a periplasmic membrane fusion protein. These tripartite machineries span both membranes and the periplasmic space, and they extrude from the bacterium chemically diverse toxic substrates. In this chapter, we summarise current understanding of the structural architecture, functionality, and regulation of tripartite multi-drug efflux assemblies.
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http://dx.doi.org/10.1016/j.resmic.2018.05.003DOI Listing
November 2018

RNase E and the High-Fidelity Orchestration of RNA Metabolism.

Microbiol Spectr 2018 04;6(2)

Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom.

The bacterial endoribonuclease RNase E occupies a pivotal position in the control of gene expression, as its actions either commit transcripts to an irreversible fate of rapid destruction or unveil their hidden functions through specific processing. Moreover, the enzyme contributes to quality control of rRNAs. The activity of RNase E can be directed and modulated by signals provided through regulatory RNAs that guide the enzyme to specific transcripts that are to be silenced. Early in its evolutionary history, RNase E acquired a natively unfolded appendage that recruits accessory proteins and RNA. These accessory factors facilitate the activity of RNase E and include helicases that remodel RNA and RNA-protein complexes, and polynucleotide phosphorylase, a relative of the archaeal and eukaryotic exosomes. RNase E also associates with enzymes from central metabolism, such as enolase and aconitase. RNase E-based complexes are diverse in composition, but generally bear mechanistic parallels with eukaryotic machinery involved in RNA-induced gene regulation and transcript quality control. That these similar processes arose independently underscores the universality of RNA-based regulation in life. Here we provide a synopsis and perspective of the contributions made by RNase E to sustain robust gene regulation with speed and accuracy.
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http://dx.doi.org/10.1128/microbiolspec.RWR-0008-2017DOI Listing
April 2018

Interplay between the catabolite repression control protein Crc, Hfq and RNA in Hfq-dependent translational regulation in Pseudomonas aeruginosa.

Nucleic Acids Res 2018 02;46(3):1470-1485

Department of Microbiology, Immunobiology and Genetics, Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, Dr. Bohrgasse 9, 1030 Vienna, Austria.

In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) act as post-transcriptional regulators during carbon catabolite repression (CCR). In this regard Crc is required for full-fledged Hfq-mediated translational repression of catabolic genes. RNAseq based transcriptome analyses revealed a significant overlap between the Crc and Hfq regulons, which in conjunction with genetic data supported a concerted action of both proteins. Biochemical and biophysical approaches further suggest that Crc and Hfq form an assembly in the presence of RNAs containing A-rich motifs, and that Crc interacts with both, Hfq and RNA. Through these interactions, Crc enhances the stability of Hfq/Crc/RNA complexes, which can explain its facilitating role in Hfq-mediated translational repression. Hence, these studies revealed for the first time insights into how an interacting protein can modulate Hfq function. Moreover, Crc is shown to interfere with binding of a regulatory RNA to Hfq, which bears implications for riboregulation. These results are discussed in terms of a working model, wherein Crc prioritizes the function of Hfq toward utilization of favored carbon sources.
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http://dx.doi.org/10.1093/nar/gkx1245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5815094PMC
February 2018

Purification of AcrAB-TolC Multidrug Efflux Pump for Cryo-EM Analysis.

Methods Mol Biol 2018 ;1700:71-81

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1GA, UK.

The cell envelope of Gram-negative bacteria comprises an outer membrane, a cytoplasmic inner membrane, and an interstitial space. The tripartite multidrug transporter AcrAB-TolC, which uses proton electrochemical gradients to vectorially drive the efflux of drugs from the cell, spans this envelope. We describe here details of the methods used to prepare the recombinant tripartite assembly for high-resolution structure determination by cryo-EM.
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http://dx.doi.org/10.1007/978-1-4939-7454-2_5DOI Listing
July 2018

Analysis of the natively unstructured RNA/protein-recognition core in the Escherichia coli RNA degradosome and its interactions with regulatory RNA/Hfq complexes.

Nucleic Acids Res 2018 01;46(1):387-402

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.

The RNA degradosome is a multi-enzyme assembly that plays a central role in the RNA metabolism of Escherichia coli and numerous other bacterial species including pathogens. At the core of the assembly is the endoribonuclease RNase E, one of the largest E. coli proteins and also one that bears the greatest region predicted to be natively unstructured. This extensive unstructured region, situated in the C-terminal half of RNase E, is punctuated with conserved short linear motifs that recruit partner proteins, direct RNA interactions, and enable association with the cytoplasmic membrane. We have structurally characterized a subassembly of the degradosome-comprising a 248-residue segment of the natively unstructured part of RNase E, the DEAD-box helicase RhlB and the glycolytic enzyme enolase, and provide evidence that it serves as a flexible recognition centre that can co-recruit small regulatory RNA and the RNA chaperone Hfq. Our results support a model in which the degradosome captures substrates and regulatory RNAs through the recognition centre, facilitates pairing to cognate transcripts and presents the target to the ribonuclease active sites of the greater assembly for cooperative degradation or processing.
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http://dx.doi.org/10.1093/nar/gkx1083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5758883PMC
January 2018

Structural insights into RapZ-mediated regulation of bacterial amino-sugar metabolism.

Nucleic Acids Res 2017 Oct;45(18):10845-10860

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.

In phylogenetically diverse bacteria, the conserved protein RapZ plays a central role in RNA-mediated regulation of amino-sugar metabolism. RapZ contributes to the control of glucosamine phosphate biogenesis by selectively presenting the regulatory small RNA GlmZ to the essential ribonuclease RNase E for inactivation. Here, we report the crystal structures of full length Escherichia coli RapZ at 3.40 Å and 3.25 Å, and its isolated C-terminal domain at 1.17 Å resolution. The structural data confirm that the N-terminal domain of RapZ possesses a kinase fold, whereas the C-terminal domain bears closest homology to a subdomain of 6-phosphofructokinase, an important enzyme in the glycolytic pathway. RapZ self-associates into a domain swapped dimer of dimers, and in vivo data support the importance of quaternary structure in RNA-mediated regulation of target gene expression. Based on biochemical, structural and genetic data, we suggest a mechanism for binding and presentation by RapZ of GlmZ and the closely related decoy sRNA, GlmY. We discuss a scenario for the molecular evolution of RapZ through re-purpose of enzyme components from central metabolism.
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http://dx.doi.org/10.1093/nar/gkx732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737377PMC
October 2017

RNA search engines empower the bacterial intranet.

Biochem Soc Trans 2017 08 14;45(4):987-997. Epub 2017 Jul 14.

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, U.K.

RNA acts not only as an information bearer in the biogenesis of proteins from genes, but also as a regulator that participates in the control of gene expression. In bacteria, small RNA molecules (sRNAs) play controlling roles in numerous processes and help to orchestrate complex regulatory networks. Such processes include cell growth and development, response to stress and metabolic change, transcription termination, cell-to-cell communication, and the launching of programmes for host invasion. All these processes require recognition of target messenger RNAs by the sRNAs. This review summarizes recent results that have provided insights into how bacterial sRNAs are recruited into effector ribonucleoprotein complexes that can seek out and act upon target transcripts. The results hint at how sRNAs and their protein partners act as pattern-matching search engines that efficaciously regulate gene expression, by performing with specificity and speed while avoiding off-target effects. The requirements for efficient searches of RNA patterns appear to be common to all domains of life.
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http://dx.doi.org/10.1042/BST20160373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5652223PMC
August 2017

1.8 Å resolution crystal structure of the carbapenem intrinsic resistance protein CarF.

Acta Crystallogr D Struct Biol 2017 Jul 22;73(Pt 7):549-556. Epub 2017 Jun 22.

Department of Biochemistry, University of Cambridge, Building O, Downing Site, Cambridge CB2 1QW, England.

The natural production of the β-lactam antibiotic carbapenem in bacteria involves a group of enzymes that form a synthetic pathway as well as proteins that protect the cell from self-intoxification by the products. Here, the crystal structure of CarF, one of the two proteins that confer resistance to synthesis of the antibiotic in the host organism, is reported. The CarF fold places it within a widely occurring structural family, indicating an ancient structural origin from which the resistance function has been derived.
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http://dx.doi.org/10.1107/S2059798317002236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610886PMC
July 2017

The self-inhibitory nature of metabolic networks and its alleviation through compartmentalization.

Nat Commun 2017 07 10;8:16018. Epub 2017 Jul 10.

Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.

Metabolites can inhibit the enzymes that generate them. To explore the general nature of metabolic self-inhibition, we surveyed enzymological data accrued from a century of experimentation and generated a genome-scale enzyme-inhibition network. Enzyme inhibition is often driven by essential metabolites, affects the majority of biochemical processes, and is executed by a structured network whose topological organization is reflecting chemical similarities that exist between metabolites. Most inhibitory interactions are competitive, emerge in the close neighbourhood of the inhibited enzymes, and result from structural similarities between substrate and inhibitors. Structural constraints also explain one-third of allosteric inhibitors, a finding rationalized by crystallographic analysis of allosterically inhibited L-lactate dehydrogenase. Our findings suggest that the primary cause of metabolic enzyme inhibition is not the evolution of regulatory metabolite-enzyme interactions, but a finite structural diversity prevalent within the metabolome. In eukaryotes, compartmentalization minimizes inevitable enzyme inhibition and alleviates constraints that self-inhibition places on metabolism.
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http://dx.doi.org/10.1038/ncomms16018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5508129PMC
July 2017

Structure of the MacAB-TolC ABC-type tripartite multidrug efflux pump.

Nat Microbiol 2017 May 15;2:17070. Epub 2017 May 15.

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, U.K.

The MacA-MacB-TolC assembly of Escherichia coli is a transmembrane machine that spans the cell envelope and actively extrudes substrates, including macrolide antibiotics and polypeptide virulence factors. These transport processes are energized by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. We present an electron cryo-microscopy structure of the ABC-type tripartite assembly at near-atomic resolution. A hexamer of the periplasmic protein MacA bridges between a TolC trimer in the outer membrane and a MacB dimer in the inner membrane, generating a quaternary structure with a central channel for substrate translocation. A gating ring found in MacA is proposed to act as a one-way valve in substrate transport. The MacB structure features an atypical transmembrane domain with a closely packed dimer interface and a periplasmic opening that is the likely portal for substrate entry from the periplasm, with subsequent displacement through an allosteric transport mechanism.
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http://dx.doi.org/10.1038/nmicrobiol.2017.70DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5447821PMC
May 2017

Association of the Cold Shock DEAD-Box RNA Helicase RhlE to the RNA Degradosome in Caulobacter crescentus.

J Bacteriol 2017 07 13;199(13). Epub 2017 Jun 13.

Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil

In diverse bacterial lineages, multienzyme assemblies have evolved that are central elements of RNA metabolism and RNA-mediated regulation. The aquatic Gram-negative bacterium , which has been a model system for studying the bacterial cell cycle, has an RNA degradosome assembly that is formed by the endoribonuclease RNase E and includes the DEAD-box RNA helicase RhlB. Immunoprecipitations of extracts from cells expressing an epitope-tagged RNase E reveal that RhlE, another member of the DEAD-box helicase family, associates with the degradosome at temperatures below those optimum for growth. Phenotype analyses of , , and mutant strains show that RhlE is important for cell fitness at low temperature and its role may not be substituted by RhlB. Transcriptional and translational fusions of to the reporter gene and immunoblot analysis of an epitope-tagged RhlE indicate that its expression is induced upon temperature decrease, mainly through posttranscriptional regulation. RNase E pulldown assays show that other proteins, including the transcription termination factor Rho, a second DEAD-box RNA helicase, and ribosomal protein S1, also associate with the degradosome at low temperature. The results suggest that the RNA degradosome assembly can be remodeled with environmental change to alter its repertoire of helicases and other accessory proteins. DEAD-box RNA helicases are often present in the RNA degradosome complex, helping unwind secondary structures to facilitate degradation. is an interesting organism to investigate degradosome remodeling with change in temperature, because it thrives in freshwater bodies and withstands low temperature. In this study, we show that at low temperature, the cold-induced DEAD-box RNA helicase RhlE is recruited to the RNA degradosome, along with other helicases and the Rho protein. RhlE is essential for bacterial fitness at low temperature, and its function may not be complemented by RhlB, although RhlE is able to complement for loss. These results suggest that RhlE has a specific role in the degradosome at low temperature, potentially improving adaptation to this condition.
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http://dx.doi.org/10.1128/JB.00135-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5472812PMC
July 2017

An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump.

Elife 2017 03 29;6. Epub 2017 Mar 29.

Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.

Bacterial efflux pumps confer multidrug resistance by transporting diverse antibiotics from the cell. In Gram-negative bacteria, some of these pumps form multi-protein assemblies that span the cell envelope. Here, we report the near-atomic resolution cryoEM structures of the AcrAB-TolC multidrug efflux pump in resting and drug transport states, revealing a quaternary structural switch that allosterically couples and synchronizes initial ligand binding with channel opening. Within the transport-activated state, the channel remains open even though the pump cycles through three distinct conformations. Collectively, our data provide a dynamic mechanism for the assembly and operation of the AcrAB-TolC pump.
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http://dx.doi.org/10.7554/eLife.24905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5404916PMC
March 2017

Structure of the ProQ RNA-binding protein.

RNA 2017 05 13;23(5):696-711. Epub 2017 Feb 13.

Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom.

The protein ProQ has recently been identified as a global small noncoding RNA-binding protein in , and a similar role is anticipated for its numerous homologs in divergent bacterial species. We report the solution structure of ProQ, revealing an N-terminal FinO-like domain, a C-terminal domain that unexpectedly has a Tudor domain fold commonly found in eukaryotes, and an elongated bridging intradomain linker that is flexible but nonetheless incompressible. Structure-based sequence analysis suggests that the Tudor domain was acquired through horizontal gene transfer and gene fusion to the ancestral FinO-like domain. Through a combination of biochemical and biophysical approaches, we have mapped putative RNA-binding surfaces on all three domains of ProQ and modeled the protein's conformation in the and RNA-bound forms. Taken together, these data suggest how the FinO, Tudor, and linker domains of ProQ cooperate to recognize complex RNA structures and serve to promote RNA-mediated regulation.
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http://dx.doi.org/10.1261/rna.060343.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393179PMC
May 2017

Structure of the ProQ RNA-binding protein.

RNA 2017 05 13;23(5):696-711. Epub 2017 Feb 13.

Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom.

The protein ProQ has recently been identified as a global small noncoding RNA-binding protein in , and a similar role is anticipated for its numerous homologs in divergent bacterial species. We report the solution structure of ProQ, revealing an N-terminal FinO-like domain, a C-terminal domain that unexpectedly has a Tudor domain fold commonly found in eukaryotes, and an elongated bridging intradomain linker that is flexible but nonetheless incompressible. Structure-based sequence analysis suggests that the Tudor domain was acquired through horizontal gene transfer and gene fusion to the ancestral FinO-like domain. Through a combination of biochemical and biophysical approaches, we have mapped putative RNA-binding surfaces on all three domains of ProQ and modeled the protein's conformation in the and RNA-bound forms. Taken together, these data suggest how the FinO, Tudor, and linker domains of ProQ cooperate to recognize complex RNA structures and serve to promote RNA-mediated regulation.
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http://dx.doi.org/10.1261/rna.060343.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393179PMC
May 2017

In Vivo Cleavage Map Illuminates the Central Role of RNase E in Coding and Non-coding RNA Pathways.

Mol Cell 2017 Jan;65(1):39-51

Institute of Molecular Infection Biology, University of Würzburg, 97080 Würzburg, Germany; Helmholtz Institute for RNA-based Infection Research (HIRI), 97080 Würzburg, Germany. Electronic address:

Understanding RNA processing and turnover requires knowledge of cleavages by major endoribonucleases within a living cell. We have employed TIER-seq (transiently inactivating an endoribonuclease followed by RNA-seq) to profile cleavage products of the essential endoribonuclease RNase E in Salmonella enterica. A dominating cleavage signature is the location of a uridine two nucleotides downstream in a single-stranded segment, which we rationalize structurally as a key recognition determinant that may favor RNase E catalysis. Our results suggest a prominent biogenesis pathway for bacterial regulatory small RNAs whereby RNase E acts together with the RNA chaperone Hfq to liberate stable 3' fragments from various precursor RNAs. Recapitulating this process in vitro, Hfq guides RNase E cleavage of a representative small-RNA precursor for interaction with a mRNA target. In vivo, the processing is required for target regulation. Our findings reveal a general maturation mechanism for a major class of post-transcriptional regulators.
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http://dx.doi.org/10.1016/j.molcel.2016.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5222698PMC
January 2017
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