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Peixuan Guo - One of the best experts on this subject based on the ideXlab platform.
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macromolecule sensing and tumor biomarker detection by harnessing terminal size and hydrophobicity of viral DNA Packaging motor channels into membranes and flow cells
Biomaterials Science, 2021Co-Authors: Long Zhang, Lakmal Jayasinghe, Michael I Jordan, Nicolas Burns, Peixuan GuoAbstract:Biological nanopores for single-pore sensing have the advantage of size homogeneity, structural reproducibility, and channel amenability. In order to translate this to clinical applications, the functional biological nanopore must be inserted into a stable system for high-throughput analysis. Here we report factors that control the rate of pore insertion into polymer membrane and analyte translocation through the channel of viral DNA Packaging motors of Phi29, T3 and T7. The hydrophobicity of aminol or carboxyl terminals and their relation to the analyte translocation were investigated. It was found that both the size and the hydrophobicity of the pore terminus are critical factors for direct membrane insertion. An N-terminus or C-terminus hydrophobic mutation is crucial for governing insertion orientation and subsequent macromolecule translocation due to the one-way traffic property. The N- or C-modification led to two different modes of application. The C-terminal insertion permits translocation of analytes such as peptides to enter the channel through the N terminus, while N-terminus insertion prevents translocation but offers the measurement of gating as a sensing parameter, thus generating a tool for detection of markers. A urokinase-type Plasminogen Activator Receptor (uPAR) binding peptide was fused into the C-terminal of Phi29 nanopore to serve as a probe for uPAR protein detection. The uPAR has proven to be a predictive biomarker in several types of cancer, including breast cancer. With an N-terminal insertion, the binding of the uPAR antigen to individual peptide probe induced discretive steps of current reduction due to the induction of channel gating. The distinctive current signatures enabled us to distinguish uPAR positive and negative tumor cell lines. This finding provides a theoretical basis for a robust biological nanopore sensing system for high-throughput macromolecular sensing and tumor biomarker detection.
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detection of single peptide with only one amino acid modification via electronic fingerprinting using reengineered durable channel of phi29 DNA Packaging motor
Biomaterials, 2021Co-Authors: Long Zhang, Michael I Jordan, Lakmal Jayasinghe, Miranda L Gardner, Julian Aldana, Nicolas Burns, Michael A Freitas, Peixuan GuoAbstract:Protein post-translational modification (PTM) is crucial to modulate protein interactions and activity in various biological processes. Emerging evidence has revealed PTM patterns participate in the pathology onset and progression of various diseases. Current PTM identification relies mainly on mass spectrometry-based approaches that limit the assessment to the entire protein population in question. Here we report a label-free method for the detection of the single peptide with only one amino acid modification via electronic fingerprinting using reengineered durable channel of phi29 DNA Packaging motor, which bears the deletion of 25-amino acids (AA) at the C-terminus or 17-AA at the internal loop of the channel. The mutant channels were used to detect propionylation modification via single-molecule fingerprinting in either the traditional patch-clamp or the portable MinION™ platform of Oxford Nanopore Technologies. Up to 2000 channels are available in the MinION™ Flow Cells. The current signatures and dwell time of individual channels were identified. Peptides with only one propionylation were differentiated. Excitingly, identification of single or multiple modifications on the MinION™ system was achieved. The successful application of PTM differentiation on the MinION™ system represents a significant advance towards developing a label-free and high-throughput detection platform utilizing nanopores for clinical diagnosis based on PTM.
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insertion of channel of phi29 DNA Packaging motor into polymer membrane for high throughput sensing
Nanomedicine: Nanotechnology Biology and Medicine, 2020Co-Authors: Michael I Jordan, Lakmal Jayasinghe, Peixuan GuoAbstract:Abstract The connector channel of bacteriophage phi29 DNA Packaging motor has been inserted into the lipid bilayer membrane and has shown potential for the sensing of DNA, RNA, chemicals, peptides, and antibodies. Properties such as high solubility and large channel size have made phi29 channel an advantageous system for those applications; however, previously studied lipid membranes have short lifetimes, and they are frangible and unstable under voltages higher than 200 mV. Thus, the application of this lipid membrane platform for clinical applications is challenging. Here we report the insertion of the connector into the stable polymer membrane in MinION flow cell that contains 2048 wells for high-throughput sensing by the liposome-polymer fusion process. The successful insertion of phi29 connector was confirmed by a unique gating phenomenon. Peptide translocation through the inserted phi29 connector was also observed, revealing the potential of applying phi29 connector for high-throughput peptide sensing.
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nano channel of viral DNA Packaging motor as single pore to differentiate peptides with single amino acid difference
Biomaterials, 2018Co-Authors: Xinqi Kang, Shaoying Wang, Peixuan GuoAbstract:Detection, differentiation, mapping, and sequencing of proteins are important in proteomics for the assessment of cell development such as protein methylation or phosphorylation as well as the diagnosis of diseases including metabolic disorder, mental illness, immunological ailments, and malignant cancers. Nanopore technology has demonstrated the potential for the sequencing or sensing of DNA, RNA, chemicals, or other macromolecules. Due to the diversity of protein in shape, structure and charge and the composition versatility of 20 amino acids, the sequencing of proteins remains challenging. Herein, we report the application of the channel of bacteriophage T7 DNA Packaging motor for the differentiation of an assortment of peptides of a single amino acid difference. Explicit fingerprints or signatures were obtained based on current blockage and dwell time of individual peptide. Data from the clear mapping of small proteins after protease digestion suggests the potential of using T7 motor channel for proteomics including protein sequencing.
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methods for construction and characterization of simple or special multifunctional rna nanoparticles based on the 3wj of phi29 DNA Packaging motor
Methods, 2018Co-Authors: Sijin Guo, Xijun Piao, Peixuan GuoAbstract:Abstract The field of RNA nanotechnology has developed rapidly over the last decade, as more elaborate RNA nanoarchitectures and therapeutic RNA nanoparticles have been constructed, and their applications have been extensively explored. Now it is time to offer different levels of RNA construction methods for both the beginners and the experienced researchers or enterprisers. The first and second parts of this article will provide instructions on basic and simple methods for the assembly and characterization of RNA nanoparticles, mainly based on the pRNA three-way junction (pRNA-3WJ) of phi29 DNA Packaging motor. The third part of this article will focus on specific methods for the construction of more sophisticated multivalent RNA nanoparticles for therapeutic applications. In these parts, some simple protocols are provided to facilitate the initiation of the RNA nanoparticle construction in labs new to the field of RNA nanotechnology. This article is intended to serve as a general reference aimed at both apprentices and senior scientists for their future design, construction and characterization of RNA nanoparticles based on the pRNA-3WJ of phi29 DNA Packaging motor.
V. B. Rao - One of the best experts on this subject based on the ideXlab platform.
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altering the speed of a DNA Packaging motor from bacteriophage t4
Nucleic Acids Research, 2017Co-Authors: Siying Lin, Tanfis I Alam, Vishal I Kottadiel, Yann R Chemla, Carl J Vangessel, Wei Chun Tang, V. B. RaoAbstract:The speed at which a molecular motor operates is critically important for the survival of a virus or an organism but very little is known about the underlying mechanisms. Tailed bacteriophage T4 employs one of the fastest and most powerful Packaging motors, a pentamer of gp17 that translocates DNA at a rate of up to ∼2000-bp/s. We hypothesize, guided by structural and genetic analyses, that a unique hydrophobic environment in the catalytic space of gp17-adenosine triphosphatase (ATPase) determines the rate at which the 'lytic water' molecule is activated and OH- nucleophile is generated, in turn determining the speed of the motor. We tested this hypothesis by identifying two hydrophobic amino acids, M195 and F259, in the catalytic space of gp17-ATPase that are in a position to modulate motor speed. Combinatorial mutagenesis demonstrated that hydrophobic substitutions were tolerated but polar or charged substitutions resulted in null or cold-sensitive/small-plaque phenotypes. Quantitative biochemical and single-molecule analyses showed that the mutant motors exhibited 1.8- to 2.5-fold lower rate of ATP hydrolysis, 2.5- to 4.5-fold lower DNA Packaging velocity, and required an activator protein, gp16 for rapid firing of ATPases. These studies uncover a speed control mechanism that might allow selection of motors with optimal performance for organisms' survival.
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mechanisms of DNA Packaging by large double stranded DNA viruses
Annual Review of Virology, 2015Co-Authors: V. B. Rao, Michael FeissAbstract:Translocation of viral double-stranded DNA (dsDNA) into the icosahedral prohead shell is catalyzed by TerL, a motor protein that has ATPase, endonuclease, and translocase activities. TerL, following endonucleolytic cleavage of immature viral DNA concatemer recognized by TerS, assembles into a pentameric ring motor on the prohead's portal vertex and uses ATP hydrolysis energy for DNA translocation. TerL's N-terminal ATPase is connected by a hinge to the C-terminal endonuclease. Inchworm models propose that modest domain motions accompanying ATP hydrolysis are amplified, through changes in electrostatic interactions, into larger movements of the C-terminal domain bound to DNA. In phage ϕ29, four of the five TerL subunits sequentially hydrolyze ATP, each powering translocation of 2.5 bp. After one viral genome is encapsidated, the internal pressure signals termination of Packaging and ejection of the motor. Current focus is on the structures of Packaging complexes and the dynamics of TerL during DNA Packaging, endonuclease regulation, and motor mechanics.
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single molecule Packaging initiation in real time by a viral DNA Packaging machine from bacteriophage t4
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Reza Vafabakhsh, Kiran Kondabagil, Zhihong Zhang, Li Dai, Tyler M Earnest, Kyung S Lee, Karin A Dahmen, V. B. RaoAbstract:Viral DNA Packaging motors are among the most powerful molecular motors known. A variety of structural, biochemical, and single-molecule biophysical approaches have been used to understand their mechanochemistry. However, Packaging initiation has been difficult to analyze because of its transient and highly dynamic nature. Here, we developed a single-molecule fluorescence assay that allowed visualization of Packaging initiation and reinitiation in real time and quantification of motor assembly and initiation kinetics. We observed that a single bacteriophage T4 Packaging machine can package multiple DNA molecules in bursts of activity separated by long pauses, suggesting that it switches between active and quiescent states. Multiple initiation pathways were discovered including, unexpectedly, direct DNA binding to the capsid portal followed by recruitment of motor subunits. Rapid succession of ATP hydrolysis was essential for efficient initiation. These observations have implications for the evolution of icosahedral viruses and regulation of virus assembly.
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Structure and function of the small terminase component of the DNA Packaging machine in T4-like bacteriophages
Proceedings of the National Academy of Sciences, 2012Co-Authors: S. Sun, Kiran Kondabagil, Michael G. Rossmann, Y Xiang, S. Gao, V. B. RaoAbstract:Tailed DNA bacteriophages assemble empty procapsids that are subsequently filled with the viral genome by means of a DNA Packaging machine situated at a special fivefold vertex. The Packaging machine consists of a "small terminase" and a "large terminase" component. One of the functions of the small terminase is to initiate Packaging of the viral genome, whereas the large terminase is responsible for the ATP-powered translocation of DNA. The small terminase subunit has three domains, an N-terminal DNA-binding domain, a central oligomerization domain, and a C-terminal domain for interacting with the large terminase. Here we report structures of the central domain in two different oligomerization states for a small terminase from the T4 family of phages. In addition, we report biochemical studies that establish the function for each of the small terminase domains. On the basis of the structural and biochemical information, we propose a model for DNA Packaging initiation.
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The bacteriophage DNA Packaging machine
Advances in Experimental Medicine and Biology, 2012Co-Authors: V. B. RaoAbstract:Large dsDNA bacteriophages and herpesviruses encode a powerful ATP-driven DNA-translocating machine that encapsidates a viral genome into a preformed capsid shell or prohead. The key components of the Packaging machine are the Packaging enzyme (terminase, motor) and the portal protein that forms the unique DNA entrance vertex of prohead. The terminase complex, comprised of a recognition subunit (small terminase) and an endonuclease/translocase subunit (large terminase), cuts viral genome concatemers. The terminase-viral DNA complex docks on the portal vertex, assembling a motor complex containing five large terminase subunits. The pentameric motor processively translocates DNA until the head shell is full with one viral genome. The motor cuts the DNA again and dissociates from the full head, allowing head-finishing proteins to assemble on the portal, sealing the portal, and constructing a platform for tail attachment. A body of evidence from molecular genetics and biochemical, structural, and biophysical approaches suggests that ATP hydrolysis-driven conformational changes in the Packaging motor (large terminase) power DNA motion. Various parts of the motor subunit, such as the ATPase, arginine finger, transmission domain, hinge, and DNA groove, work in concert to translocate about 2 bp of DNA per ATP hydrolyzed. Powerful single-molecule approaches are providing precise delineation of steps during each translocation event in a motor that has a speed as high as a millisecond/step. The phage Packaging machine has emerged as an excellent model for understanding the molecular machines, given the mechanistic parallels between terminases, helicases, and numerous motor proteins.
D Smith - One of the best experts on this subject based on the ideXlab platform.
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evidence that a catalytic glutamate and an arginine toggle act in concert to mediate atp hydrolysis and mechanochemical coupling in a viral DNA Packaging motor
Nucleic Acids Research, 2019Co-Authors: David Ortiz, Damian Deltoro, Mariam Ordyan, Joshua Pajak, Jean Sippy, Alexis Catala, Gaurav Arya, Michael Feiss, D Smith, Carlos Enrique CatalanoAbstract:ASCE ATPases include ring-translocases such as cellular helicases and viral DNA Packaging motors (terminases). These motors have conserved Walker A and B motifs that bind Mg2+-ATP and a catalytic carboxylate that activates water for hydrolysis. Here we demonstrate that Glu179 serves as the catalytic carboxylate in bacteriophage λ terminase and probe its mechanistic role. All changes of Glu179 are lethal: non-conservative changes abrogate ATP hydrolysis and DNA translocation, while the conservative E179D change attenuates ATP hydrolysis and alters single molecule translocation dynamics, consistent with a slowed chemical hydrolysis step. Molecular dynamics simulations of several homologous terminases suggest a novel mechanism, supported by experiments, wherein the conserved Walker A arginine 'toggles' between interacting with a glutamate residue in the 'lid' subdomain and the catalytic glutamate upon ATP binding; this switch helps mediate a transition from an 'open' state to a 'closed' state that tightly binds nucleotide and DNA, and also positions the catalytic glutamate next to the γ-phosphate to align the hydrolysis transition state. Concomitant reorientation of the lid subdomain may mediate mechanochemical coupling of ATP hydrolysis and DNA translocation. Given the strong conservation of these structural elements in terminase enzymes, this mechanism may be universal for viral Packaging motors.
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single molecule measurements of motor driven viral DNA Packaging in bacteriophages phi29 lambda and t4 with optical tweezers
Methods of Molecular Biology, 2018Co-Authors: Nick Keller, Damian Deltoro, D SmithAbstract:Viral DNA Packaging is a required step in the assembly of many dsDNA viruses. A molecular motor fueled by ATP hydrolysis packages the viral genome to near crystalline density inside a preformed prohead shell in ~5 min at room temperature. We describe procedures for measuring the Packaging of single DNA molecules into single viral proheads with optical tweezers. Three viral Packaging systems are described in detail: bacteriophages phi29 (φ29), lambda (λ), and T4. Two different approaches are described: (1) With φ29 and T4, prohead-motor complexes can be preassembled in bulk and Packaging can be initiated in the optical tweezers by "feeding" a single DNA molecule to one of the complexes; (2) With φ29 and λ, Packaging can be initiated in bulk then stalled, and a single prohead-motor-DNA complex can then be captured with optical tweezers and restarted. In both cases, the prohead is ultimately attached to one trapped microsphere and the end of the DNA being packaged is attached to a second trapped microsphere such that Packaging of the DNA pulls the two microspheres together and the rate of Packaging and force generated by the motor is directly measured in real time. These protocols allow for the effect of many experimental parameters on Packaging dynamics to be studied such as temperature, ATP concentration, ionic conditions, structural changes to the DNA substrate, and mutations in the motor proteins. Procedures for capturing microspheres with the optical traps and different measurement modes are also described.
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walker a motif acts to coordinate atp hydrolysis with motor output in viral DNA Packaging
Journal of Molecular Biology, 2016Co-Authors: Damian Deltoro, David Ortiz, Mariam Ordyan, Jean Sippy, Carlos Enrique Catalano, Michael Feiss, Nick Keller, D SmithAbstract:During the assembly of many viruses, a powerful ATP-driven motor translocates DNA into a preformed procapsid. A Walker-A "P-loop" motif is proposed to coordinate ATP binding and hydrolysis with DNA translocation. We use genetic, biochemical, and biophysical techniques to survey the roles of P-loop residues in bacteriophage lambda motor function. We identify 55 point mutations that reduce virus yield to below detectable levels in a highly sensitive genetic complementation assay and 33 that cause varying reductions in yield. Most changes in the predicted conserved residues K76, R79, G81, and S83 produce no detectable yield. Biochemical analyses show that R79A and S83A mutant proteins fold, assemble, and display genome maturation activity similar to wild-type (WT) but exhibit little ATPase or DNA Packaging activity. Kinetic DNA cleavage and ATPase measurements implicate R79 in motor ring assembly on DNA, supporting recent structural models that locate the P-loop at the interface between motor subunits. Single-molecule measurements detect no translocation for K76A and K76R, while G81A and S83A exhibit strong impairments, consistent with their predicted roles in ATP binding. We identify eight residue changes spanning A78-K84 that yield impaired translocation phenotypes and show that Walker-A residues play important roles in determining motor velocity, pausing, and processivity. The efficiency of initiation of Packaging correlates strongly with motor velocity. Frequent pausing and slipping caused by changes A78V and R79K suggest that these residues are important for ATP alignment and coupling of ATP binding to DNA gripping. Our findings support recent structural models implicating the P-loop arginine in ATP hydrolysis and mechanochemical coupling.
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evidence for an electrostatic mechanism of force generation by the bacteriophage t4 DNA Packaging motor
Nature Communications, 2014Co-Authors: Amy Migliori, Gaurav Arya, V.basaveswara Rao, Nick Keller, Tanfis I Alam, Marthandan Mahalingam, D SmithAbstract:Viral DNA Packaging motors must generate large forces to package the viral capsid. Here, Migliori et al. provide functional and computational evidence that electrostatic interactions between subdomains of the T4 Packaging motor provide the driving force for DNA Packaging.
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probing functional motifs and regions in the phage lambda DNA Packaging motor by mutagenesis and single molecule optical tweezers measurements
Biophysical Journal, 2012Co-Authors: D SmithAbstract:A key step in the assembly of many viruses is the Packaging of DNA into preformed procapsids by an ATP-powered molecular motor. To shed light on the motor mechanism we used optical tweezers measurements to study the effect on DNA translocation dynamics of amino acid changes in the large terminase subunit (gpA) of the phage lambda Packaging motor. Observed changes in motor velocity, processivity, and/or velocity-force dependence provide evidence supporting the assignment of a Walker A-like phosphate-binding motif, an adenine binding Q motif analogous to that recently identified in RNA helicases, and a C motif that couples ATP hydrolysis to DNA translocation. In addition, we found that a residue change T194M in a predicted loop-helix-loop region outside any previously described motifs, caused a dramatic 8-fold reduction in motor velocity without changing processivity or force-dependence. T194 lies in a loop-helix-loop region that is predicted to position key residues of Walker B and C catalytic motifs and appears to regulate DNA translocation rate in both the lambda DNA Packaging motor and the homologous B. Subtilis SpoIIIE chromosome segregation motor.
Shelley Grimes - One of the best experts on this subject based on the ideXlab platform.
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an rna domain imparts specificity and selectivity to a viral DNA Packaging motor
Journal of Virology, 2015Co-Authors: Wei Zhao, Paul J. Jardine, Shelley GrimesAbstract:UNLABELLED During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the Packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the Packaging process. DNA Packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA Packaging, selectively Packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, Packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the Packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. IMPORTANCE During virus assembly, genome Packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA Packaging is recognition of the viral DNA among other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage ϕ29, this essential step of genome recognition is mediated by a viral genome-encoded RNA rather than a protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA Packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA Packaging is a novel target for the development of antiviral drugs.
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Dye Labeling and DNA Packaging of φ29 Proheads
2013Co-Authors: Thorsten Hugel, Paul J. Jardine, Shelley Grimes, Dwight L. Anderson, Craig L. Hetherington, Jens Michaelis, Jessica M Walter, Wayne Falk, Carlos BustamanteAbstract:(A) SDS-PAGE of 170C-connector-mutant proheads. Protein stain of proheads shows the structural components gp8 (capsid), gp8.5 (fiber), gp10 (connector), and gp7 (scaffold) in lane (a). Fluorescence scan of the gel showing labeled proheads with various amounts of dye per gp10 monomer used in labeling reaction: 1 dye per gp10, lane (b); 0.5 dyes per gp10, lane (c); 0.25 dyes per gp10, lane (d); 0.125 dyes per gp10, lane (e); 0.0625 dyes per gp10, lane (f); and no dye, lane (g). The bands in the fluorescence scan with no match in the protein stain originate from highly reactive but quantitatively minor E. coli proteins. (B) DNA Packaging tested by nuclease (EcoRI) protection assay using the labeled proheads from (A). Lane (a) shows input DNA-gp3; lane (b) shows a negative (no ATP) control. Packaged DNA is protected from nuclease digestion. Packaging activity is unaffected by dye labeling when compared to a 193C Packaging control, lane (c). (B) Shows labeled proheads from (A), ranging from 1 dye per gp10, lane (d); 0.5 dyes per gp10, lane (e); through to no dye, lane (i).
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Structure of the RNA claw of the DNA Packaging motor of bacteriophage φ29
Nucleic acids research, 2012Co-Authors: Elena Harjes, Paul J. Jardine, Marc C. Morais, Shelley Grimes, Wei Zhao, Aya Kitamura, Hiroshi MatsuoAbstract:Bacteriophage DNA Packaging motors translocate their genomic DNA into viral heads, compacting it to near-crystalline density. The Bacillus subtilis phage 29 has a unique ring of RNA (pRNA) that is an essential component of its motor, serving as a scaffold for the Packaging ATPase. Previously, deletion of a three-base bulge (18-CCA-20) in the pRNA A-helix was shown to abolish Packaging activity. Here, we solved the structure of this crucial bulge by nuclear magnetic resonance (NMR) using a 27mer RNA fragment containing the bulge (27b). The bulge actually involves five nucleotides (17-UCCA-20 and A100), as U17 and A100 are not base paired as predicted. Mutational analysis showed these newly identified bulge residues are important for DNA Packaging. The bulge introduces a 33-35° bend in the helical axis, and inter-helical motion around this bend appears to be restricted. A model of the functional 120b pRNA was generated using a 27b NMR structure and the crystal structure of the 66b prohead-binding domain. Fitting this model into a cryo-EM map generated a pentameric pRNA structure; five helices projecting from the pRNA ring resemble an RNA claw. Biochemical analysis suggested that this shape is important for coordinated motor action required for DNA translocation.
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Role of φ29 connector channel loops in late-stage DNA Packaging.
Journal of molecular biology, 2011Co-Authors: Shelley Grimes, Paul J. Jardine, Jiali Gao, Rockney AtzAbstract:Abstract Double-stranded DNA bacteriophages and their eukaryotic virus counterparts have 12-fold head–tail connector assemblages embedded at a unique capsid vertex. This vertex is the site of assembly of the DNA Packaging motor, and the connector has a central channel through which viral DNA passes during genome Packaging and subsequent host infection. Crystal structures of connectors from different phages reveal either disordered residues or structured loops that project into the connector channel. Given the proximity to the translocating DNA substrate, these loops have been proposed to play a role in DNA Packaging. Previous models have proposed structural motions in either the Packaging ATPase or the connector channel loops as the driving force that translocates the DNA into the prohead. Here, we mutate the channel loops of the Bacillus subtilis bacteriophage φ29 connector and show that these loops have no active role in translocation of DNA. Instead, they appear to have an essential function near the end of Packaging, acting to retain the packaged DNA in the head in preparation for motor detachment and subsequent tail assembly and virion completion.
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Structure and assembly of the essential RNA ring component of a viral DNA Packaging motor
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Paul J. Jardine, Shelley Grimes, Dwight L. Anderson, Wei Zhao, Fang Ding, Kanagalaghatta R. RajashankarAbstract:Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage 29 DNA Packaging motor. The pRNA forms a multimeric ring via intermolecular base-pairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA Packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 Å resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNA cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA Packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the Packaging of 29 DNA.
Paul J. Jardine - One of the best experts on this subject based on the ideXlab platform.
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Experimental comparison of forces resisting viral DNA Packaging and driving DNA ejection
Physical Review E, 2017Co-Authors: Nicholas Keller, Paul J. Jardine, Zachary T Berndsen, Douglas E. SmithAbstract:We compare forces resisting DNA Packaging and forces driving DNA ejection in bacteriophage phi29 with theoretical predictions. Ejection of DNA from prohead-motor complexes is triggered by heating complexes after in vitro Packaging and force is inferred from the suppression of ejection by applied osmotic pressure. Ejection force from $0%$ to $80%$ filling is found to be in quantitative agreement with predictions of a continuum mechanics model that assumes a repulsive DNA-DNA interaction potential based on DNA condensation studies and predicts an inverse-spool conformation. Force resisting DNA Packaging from $\ensuremath{\sim}80%$ to $100%$ filling inferred from optical tweezers studies is also consistent with the predictions of this model. The striking agreement with these two different measurements suggests that the overall energetics of DNA Packaging is well described by the model. However, since electron microscopy studies of phi29 do not reveal a spool conformation, our findings suggest that the spool model overestimates the role of bending rigidity and underestimates the role of intrastrand repulsion. Below $\ensuremath{\sim}80%$ filling the inferred forces resisting Packaging are unexpectedly lower than the inferred ejection forces, suggesting that in this filling range the forces are less accurately determined or strongly temperature dependent.
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an rna domain imparts specificity and selectivity to a viral DNA Packaging motor
Journal of Virology, 2015Co-Authors: Wei Zhao, Paul J. Jardine, Shelley GrimesAbstract:UNLABELLED During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the Packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the Packaging process. DNA Packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA Packaging, selectively Packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, Packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the Packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. IMPORTANCE During virus assembly, genome Packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA Packaging is recognition of the viral DNA among other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage ϕ29, this essential step of genome recognition is mediated by a viral genome-encoded RNA rather than a protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA Packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA Packaging is a novel target for the development of antiviral drugs.
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Dye Labeling and DNA Packaging of φ29 Proheads
2013Co-Authors: Thorsten Hugel, Paul J. Jardine, Shelley Grimes, Dwight L. Anderson, Craig L. Hetherington, Jens Michaelis, Jessica M Walter, Wayne Falk, Carlos BustamanteAbstract:(A) SDS-PAGE of 170C-connector-mutant proheads. Protein stain of proheads shows the structural components gp8 (capsid), gp8.5 (fiber), gp10 (connector), and gp7 (scaffold) in lane (a). Fluorescence scan of the gel showing labeled proheads with various amounts of dye per gp10 monomer used in labeling reaction: 1 dye per gp10, lane (b); 0.5 dyes per gp10, lane (c); 0.25 dyes per gp10, lane (d); 0.125 dyes per gp10, lane (e); 0.0625 dyes per gp10, lane (f); and no dye, lane (g). The bands in the fluorescence scan with no match in the protein stain originate from highly reactive but quantitatively minor E. coli proteins. (B) DNA Packaging tested by nuclease (EcoRI) protection assay using the labeled proheads from (A). Lane (a) shows input DNA-gp3; lane (b) shows a negative (no ATP) control. Packaged DNA is protected from nuclease digestion. Packaging activity is unaffected by dye labeling when compared to a 193C Packaging control, lane (c). (B) Shows labeled proheads from (A), ranging from 1 dye per gp10, lane (d); 0.5 dyes per gp10, lane (e); through to no dye, lane (i).
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Structure of the RNA claw of the DNA Packaging motor of bacteriophage φ29
Nucleic acids research, 2012Co-Authors: Elena Harjes, Paul J. Jardine, Marc C. Morais, Shelley Grimes, Wei Zhao, Aya Kitamura, Hiroshi MatsuoAbstract:Bacteriophage DNA Packaging motors translocate their genomic DNA into viral heads, compacting it to near-crystalline density. The Bacillus subtilis phage 29 has a unique ring of RNA (pRNA) that is an essential component of its motor, serving as a scaffold for the Packaging ATPase. Previously, deletion of a three-base bulge (18-CCA-20) in the pRNA A-helix was shown to abolish Packaging activity. Here, we solved the structure of this crucial bulge by nuclear magnetic resonance (NMR) using a 27mer RNA fragment containing the bulge (27b). The bulge actually involves five nucleotides (17-UCCA-20 and A100), as U17 and A100 are not base paired as predicted. Mutational analysis showed these newly identified bulge residues are important for DNA Packaging. The bulge introduces a 33-35° bend in the helical axis, and inter-helical motion around this bend appears to be restricted. A model of the functional 120b pRNA was generated using a 27b NMR structure and the crystal structure of the 66b prohead-binding domain. Fitting this model into a cryo-EM map generated a pentameric pRNA structure; five helices projecting from the pRNA ring resemble an RNA claw. Biochemical analysis suggested that this shape is important for coordinated motor action required for DNA translocation.
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Role of φ29 connector channel loops in late-stage DNA Packaging.
Journal of molecular biology, 2011Co-Authors: Shelley Grimes, Paul J. Jardine, Jiali Gao, Rockney AtzAbstract:Abstract Double-stranded DNA bacteriophages and their eukaryotic virus counterparts have 12-fold head–tail connector assemblages embedded at a unique capsid vertex. This vertex is the site of assembly of the DNA Packaging motor, and the connector has a central channel through which viral DNA passes during genome Packaging and subsequent host infection. Crystal structures of connectors from different phages reveal either disordered residues or structured loops that project into the connector channel. Given the proximity to the translocating DNA substrate, these loops have been proposed to play a role in DNA Packaging. Previous models have proposed structural motions in either the Packaging ATPase or the connector channel loops as the driving force that translocates the DNA into the prohead. Here, we mutate the channel loops of the Bacillus subtilis bacteriophage φ29 connector and show that these loops have no active role in translocation of DNA. Instead, they appear to have an essential function near the end of Packaging, acting to retain the packaged DNA in the head in preparation for motor detachment and subsequent tail assembly and virion completion.