Structural Basis of Mitochondrial Transcription Initiation.

Transcription in human mitochondria is driven by a single-subunit, factor-dependent RNA polymerase (mtRNAP). Despite its critical role in both expression and replication of the mitochondrial genome, transcription initiation by mtRNAP remains poorly understood. Here, we report crystal structures of human mitochondrial transcription initiation complexes assembled on both light and heavy strand promoters. The structures reveal how transcription factors TFAM and TFB2M assist mtRNAP to achieve promoter-dependent initiation. TFAM tethers the N-terminal region of mtRNAP to recruit the polymerase to the promoter whereas TFB2M induces structural changes in mtRNAP to enable promoter opening and trapping of the DNA non-template strand. Structural comparisons demonstrate that the initiation mechanism in mitochondria is distinct from that in the well-studied nuclear, bacterial, or bacteriophage transcription systems but that similarities are found on the topological and conceptual level. These results provide a framework for studying the regulation of gene expression and DNA replication in mitochondria.

It seems like only yesterday.

I spent my childhood and adolescence in North and South Carolina, attended Duke University, and then entered Duke Medical School. One year in the laboratory of George Schwert in the biochemistry department kindled my interest in biochemistry. After one year of residency on the medical service of Duke Hospital, chaired by Eugene Stead, I joined the group of Arthur Kornberg at Stanford Medical School as a postdoctoral fellow. Two years later I accepted a faculty position at Harvard Medical School, where I remain today. During these 50 years, together with an outstanding group of students, postdoctoral fellows, and collaborators, I have pursued studies on DNA replication. I have experienced the excitement of discovering a number of important enzymes in DNA replication that, in turn, triggered an interest in the dynamics of a replisome. My associations with industry have been stimulating and fostered new friendships. I could not have chosen a better career.

Shutoff of host transcription triggers a toxin-antitoxin system to cleave phage RNA and abort infection.

Toxin-antitoxin (TA) systems are widespread in bacteria, but their activation mechanisms and bona fide targets remain largely unknown. Here, we characterize a type III TA system, toxIN, that protects E. coli against multiple bacteriophages, including T4. Using RNA sequencing, we find that the endoribonuclease ToxN is activated following T4 infection and blocks phage development primarily by cleaving viral mRNAs and inhibiting their translation. ToxN activation arises from T4-induced shutoff of host transcription, specifically of toxIN, leading to loss of the intrinsically unstable toxI antitoxin. Transcriptional shutoff is necessary and sufficient for ToxN activation. Notably, toxIN does not strongly protect against another phage, T7, which incompletely blocks host transcription. Thus, our results reveal a critical trade-off in blocking host transcription: it helps phage commandeer host resources but can activate potent defense systems. More generally, our results now reveal the native targets of an RNase toxin and activation mechanism of a phage-defensive TA system.

Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution.

Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 Å resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 Å hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a ∼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysis, DNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.

Prokaryotic viperins produce diverse antiviral molecules.

Viperin is an interferon-induced cellular protein that is conserved in animals1. It has previously been shown to inhibit the replication of multiple viruses by producing the ribonucleotide 3'-deoxy-3',4'-didehydro (ddh)-cytidine triphosphate (ddhCTP), which acts as a chain terminator for viral RNA polymerase2. Here we show that eukaryotic viperin originated from a clade of bacterial and archaeal proteins that protect against phage infection. Prokaryotic viperins produce a set of modified ribonucleotides that include ddhCTP, ddh-guanosine triphosphate (ddhGTP) and ddh-uridine triphosphate (ddhUTP). We further show that prokaryotic viperins protect against T7 phage infection by inhibiting viral polymerase-dependent transcription, suggesting that it has an antiviral mechanism of action similar to that of animal viperin. Our results reveal a class of potential natural antiviral compounds produced by bacterial immune systems.

Definition of the binding specificity of the T7 bacteriophage primase by analysis of a protein binding microarray using a thermodynamic model.

Protein binding microarrays (PBM), SELEX, RNAcompete and chromatin-immunoprecipitation have been intensively used to determine the specificity of nucleic acid binding proteins. While the specificity of proteins with pronounced sequence specificity is straightforward, the determination of the sequence specificity of proteins of modest sequence specificity is more difficult. In this work, an explorative data analysis workflow for nucleic acid binding data was developed that can be used by scientists that want to analyse their binding data. The workflow is based on a regressor realized in scikit-learn, the major machine learning module for the scripting language Python. The regressor is built on a thermodynamic model of nucleic acid binding and describes the sequence specificity with base- and position-specific energies. The regressor was used to determine the binding specificity of the T7 primase. For this, we reanalysed the binding data of the T7 primase obtained with a custom PBM. The binding specificity of the T7 primase agrees with the priming specificity (5'-GTC) and the template (5'-GGGTC) for the preferentially synthesized tetraribonucleotide primer (5'-pppACCC) but is more relaxed. The dominant contribution of two positions in the motif can be explained by the involvement of the initiating and elongating nucleotides for template binding.

DNA-terminus-dependent transcription by T7 RNA polymerase and its C-helix mutants.

The remarkable success of messenger RNA (mRNA)-based vaccines has underscored their potential as a novel biotechnology platform for vaccine development and therapeutic protein delivery. However, the single-subunit RNA polymerase from bacteriophage T7 widely used for in vitro transcription is well known to generate double-stranded RNA (dsRNA) by-products that strongly stimulate the mammalian innate immune response. The dsRNA was reported to be originated from self-templated RNA extension or promoter-independent transcription. Here, we identified that the primary source of the full-length dsRNA during in vitro transcription is the DNA-terminus-initiated transcription by T7 RNA polymerase. Guanosines or cytosines at the end of DNA templates enhance the DNA-terminus-initiated transcription. Moreover, we found that aromatic residues located at position 47 in the C-helix lead to a significant reduction in the production of full-length dsRNA. As a result, the mRNA synthesized using the T7 RNA polymerase G47W mutant exhibits higher expression efficiency and lower immunogenicity compared to the mRNA produced using the wild-type T7 RNA polymerase.

Ocr-mediated suppression of BrxX unveils a phage counter-defense mechanism.

The burgeoning crisis of antibiotic resistance has directed attention to bacteriophages as natural antibacterial agents capable of circumventing bacterial defenses. Central to this are the bacterial defense mechanisms, such as the BREX system, which utilizes the methyltransferase BrxX to protect against phage infection. This study presents the first in vitro characterization of BrxX from Escherichia coli, revealing its substrate-specific recognition and catalytic activity. We demonstrate that BrxX exhibits nonspecific DNA binding but selectively methylates adenine within specific motifs. Kinetic analysis indicates a potential regulation of BrxX by the concentration of its co-substrate, S-adenosylmethionine, and suggests a role for other BREX components in modulating BrxX activity. Furthermore, we elucidate the molecular mechanism by which the T7 phage protein Ocr (Overcoming classical restriction) inhibits BrxX. Despite low sequence homology between BrxX from different bacterial species, Ocr effectively suppresses BrxX's enzymatic activity through high-affinity binding. Cryo-electron microscopy and biophysical analyses reveal that Ocr, a DNA mimic, forms a stable complex with BrxX, highlighting a conserved interaction interface across diverse BrxX variants. Our findings provide insights into the strategic counteraction by phages against bacterial defense systems and offer a foundational understanding of the complex interplay between phages and their bacterial hosts, with implications for the development of phage therapy to combat antibiotic resistance.

Single-mode termination of phage transcriptions, disclosing bacterial adaptation for facilitated reinitiations.

Bacterial and bacteriophage RNA polymerases (RNAPs) have divergently evolved and share the RNA hairpin-dependent intrinsic termination of transcription. Here, we examined phage T7, T3 and SP6 RNAP terminations utilizing the single-molecule fluorescence assays we had developed for bacterial terminations. We discovered the phage termination mode or outcome is virtually single with decomposing termination. Therein, RNAP is displaced forward along DNA and departs both RNA and DNA for one-step decomposition, three-dimensional diffusion and reinitiation at any promoter. This phage displacement-mediated decomposing termination is much slower than readthrough and appears homologous with the bacterial one. However, the phage sole mode of termination contrasts with the bacterial dual mode, where both decomposing and recycling terminations occur compatibly at any single hairpin- or Rho-dependent terminator. In the bacterial recycling termination, RNA is sheared from RNA·DNA hybrid, and RNAP remains bound to DNA for one-dimensional diffusion, which enables facilitated recycling for reinitiation at the nearest promoter located downstream or upstream in the sense or antisense orientation. Aligning with proximity of most terminators to adjacent promoters in bacterial genomes, the shearing-mediated recycling termination could be bacterial adaptation for the facilitated reinitiations repeated at a promoter for accelerated expression and coupled at adjoining promoters for coordinated regulation.

Extending the Host Range of Bacteriophage Particles for DNA Transduction.

A major limitation in using bacteriophage-based applications is their narrow host range. Approaches for extending the host range have focused primarily on lytic phages in hosts supporting their propagation rather than approaches for extending the ability of DNA transduction into phage-restrictive hosts. To extend the host range of T7 phage for DNA transduction, we have designed hybrid particles displaying various phage tail/tail fiber proteins. These modular particles were programmed to package and transduce DNA into hosts that restrict T7 phage propagation. We have also developed an innovative generalizable platform that considerably enhances DNA transfer into new hosts by artificially selecting tails that efficiently transduce DNA. In addition, we have demonstrated that the hybrid particles transduce desired DNA into desired hosts. This study thus critically extends and improves the ability of the particles to transduce DNA into novel phage-restrictive hosts, providing a platform for myriad applications that require this ability.

Regulation of T7 gp2.5 binding dynamics by its C-terminal tail, template conformation and sequence.

Bacteriophage T7 single-stranded DNA-binding protein (gp2.5) binds to and protects transiently exposed regions of single-stranded DNA (ssDNA) while dynamically interacting with other proteins of the replication complex. We directly visualize fluorescently labelled T7 gp2.5 binding to ssDNA at the single-molecule level. Upon binding, T7 gp2.5 reduces the contour length of ssDNA by stacking nucleotides in a force-dependent manner, suggesting T7 gp2.5 suppresses the formation of secondary structure. Next, we investigate the binding dynamics of T7 gp2.5 and a deletion mutant lacking 21 C-terminal residues (gp2.5-Δ21C) under various template tensions. Our results show that the base sequence of the DNA molecule, ssDNA conformation induced by template tension, and the acidic terminal domain from T7 gp2.5 significantly impact on the DNA binding parameters of T7 gp2.5. Moreover, we uncover a unique template-catalyzed recycling behaviour of T7 gp2.5, resulting in an apparent cooperative binding to ssDNA, facilitating efficient spatial redistribution of T7 gp2.5 during the synthesis of successive Okazaki fragments. Overall, our findings reveal an efficient binding mechanism that prevents the formation of secondary structures by enabling T7 gp2.5 to rapidly rebind to nearby exposed ssDNA regions, during lagging strand DNA synthesis.

Computationally exploring the mechanism of bacteriophage T7 gp4 helicase translocating along ssDNA.

Bacteriophage T7 gp4 helicase has served as a model system for understanding mechanisms of hexameric replicative helicase translocation. The mechanistic basis of how nucleoside 5'-triphosphate hydrolysis and translocation of gp4 helicase are coupled is not fully resolved. Here, we used a thermodynamically benchmarked coarse-grained protein force field, Associative memory, Water mediated, Structure and Energy Model (AWSEM), with the single-stranded DNA (ssDNA) force field 3SPN.2C to investigate gp4 translocation. We found that the adenosine 5'-triphosphate (ATP) at the subunit interface stabilizes the subunit-subunit interaction and inhibits subunit translocation. Hydrolysis of ATP to adenosine 5'-diphosphate enables the translocation of one subunit, and new ATP binding at the new subunit interface finalizes the subunit translocation. The LoopD2 and the N-terminal primase domain provide transient protein-protein and protein-DNA interactions that facilitate the large-scale subunit movement. The simulations of gp4 helicase both validate our coarse-grained protein-ssDNA force field and elucidate the molecular basis of replicative helicase translocation.

Mechanism by which T7 bacteriophage protein Gp1.2 inhibits Escherichia coli dGTPase.

Levels of the cellular dNTPs, the direct precursors for DNA synthesis, are important for DNA replication fidelity, cell cycle control, and resistance against viruses. Escherichia coli encodes a dGTPase (2'-deoxyguanosine-5'-triphosphate [dGTP] triphosphohydrolase [dGTPase]; dgt gene, Dgt) that establishes the normal dGTP level required for accurate DNA replication but also plays a role in protecting E. coli against bacteriophage T7 infection by limiting the dGTP required for viral DNA replication. T7 counteracts Dgt using an inhibitor, the gene 1.2 product (Gp1.2). This interaction is a useful model system for studying the ongoing evolutionary virus/host "arms race." We determined the structure of Gp1.2 by NMR spectroscopy and solved high-resolution cryo-electron microscopy structures of the Dgt-Gp1.2 complex also including either dGTP substrate or GTP coinhibitor bound in the active site. These structures reveal the mechanism by which Gp1.2 inhibits Dgt and indicate that Gp1.2 preferentially binds the GTP-bound form of Dgt. Biochemical assays reveal that the two inhibitors use different modes of inhibition and bind to Dgt in combination to yield enhanced inhibition. We thus propose an in vivo inhibition model wherein the Dgt-Gp1.2 complex equilibrates with GTP to fully inactivate Dgt, limiting dGTP hydrolysis and preserving the dGTP pool for viral DNA replication.

Motors, switches, and contacts in the replisome.

Replisomes are the protein assemblies that replicate DNA. They function as molecular motors to catalyze template-mediated polymerization of nucleotides, unwinding of DNA, the synthesis of RNA primers, and the assembly of proteins on DNA. The replisome of bacteriophage T7 contains a minimum of proteins, thus facilitating its study. This review describes the molecular motors and coordination of their activities, with emphasis on the T7 replisome. Nucleotide selection, movement of the polymerase, binding of the processivity factor, unwinding of DNA, and RNA primer synthesis all require conformational changes and protein contacts. Lagging-strand synthesis is mediated via a replication loop whose formation and resolution is dictated by switches to yield Okazaki fragments of discrete size. Both strands are synthesized at identical rates, controlled by a molecular brake that halts leading-strand synthesis during primer synthesis. The helicase serves as a reservoir for polymerases that can initiate DNA synthesis at the replication fork. We comment on the differences in other systems where applicable.

Excessive excision of correct nucleotides during DNA synthesis explained by replication hurdles.

The proofreading exonuclease activity of replicative DNA polymerase excises misincorporated nucleotides during DNA synthesis, but these events are rare. Therefore, we were surprised to find that T7 replisome excised nearly 7% of correctly incorporated nucleotides during leading and lagging strand syntheses. Similar observations with two other DNA polymerases establish its generality. We show that excessive excision of correctly incorporated nucleotides is not due to events such as processive degradation of nascent DNA or spontaneous partitioning of primer-end to the exonuclease site as a "cost of proofreading". Instead, we show that replication hurdles, including secondary structures in template, slowed helicase, or uncoupled helicase-polymerase, increase DNA reannealing and polymerase backtracking, and generate frayed primer-ends that are shuttled to the exonuclease site and excised efficiently. Our studies indicate that active-site shuttling occurs at a high frequency, and we propose that it serves as a proofreading mechanism to protect primer-ends from mutagenic extensions.

A method for rapid and reliable quantification of VEGF-cell binding activity.

Vascular endothelial growth factor (VEGF) is a pleiotropic growth factor that binds a broad spectrum of cell types and regulates diverse cellular processes, including angiogenesis, growth and survival. However, it is technically difficult to quantify VEGF-cell binding activity because of reversible nature of ligand-receptor interactions. Here we used T7 bacteriophage display to quantify and compare binding activity of three human VEGF-A (hVEGF) isoforms, including hVEGF111, 165 and 206. All three isoforms bound equally well to immobilized aflibercept, a decoy VEGF receptor. hVEGF111-Phage exhibited minimal binding to immobilized heparan sulfate, whereas hVEGF206-Phage and hVEGF165-Phage had the highest and intermediate binding to heparan, respectively. In vitro studies revealed that all three isoforms bound to human umbilical vein endothelial cells (HUVECs), HEK293 epithelial and SK-N-AS neuronal cells. hVEGF111-Phage has the lowest binding activity, while hVEGF206-Phage has the highest binding. hVEGF206-Phage was the most sensitive to detect VEGF-cell binding, albeit with the highest background binding to SK-N-AS cells. These results suggest that hVEGF206-Phage is the best-suited isoform to quantify VEGF-cell binding even though VEGF165 is the most biologically active. Furthermore, this study demonstrates the utility of T7 phage display as a platform for rapid and convenient ligand-cell binding quantification with pros and cons discussed.

Analyzing the impact of T7L variants overexpression on the metabolic profile of Escherichia coli.

INTRODUCTION: Exploring metabolic changes within host E. coli through an untargeted metabolomic study of T7L variants overexpression to optimize engineered endolysins for clinical/therapeutic use. AIM AND OBJECTIVE: This study aims to assess the impact of overexpressing T7L variants on the metabolic profiles of E. coli. The two variants considered include T7L-H37A, which has enhanced lytic activity compared to its wild-type protein, and T7L-H48K, a dead mutant with no significant activity. METHODS: 1H NMR-based metabolomics was employed to compare the metabolic profiles of E. coli cells overexpressing T7L wild-type protein and its variants. RESULTS: Overexpression of the T7L wild-type (T7L-WT) protein and its variants (T7L-H48K and T7L-H37A) was compared to RNAP overexpression in E. coli cells using 1H NMR-based metabolomics, analyzing a total of 75 annotated metabolites, including organic acids, amino acids, sugars, and nucleic acids. The results showed distinct clustering patterns for the two T7L variant groups compared with the WT, in which the dead mutant (H48K) group showed clustering close to that of RNAP. Pathway impact analysis revealed different effects of T7L variants on E. coli metabolic profiles, with T7L-H48K showing minimal alterations in energy and amino acid pathways linked to osmotic stress compared to noticeable alterations in these pathways for both T7L-H37A and T7L-WT. CONCLUSIONS: This study uncovered distinct metabolic fingerprints when comparing the overexpression of active and inactive mutants of T7L lytic enzymes in E. coli cells. These findings could contribute to the optimization and enhancement of suitable endolysins as potential alternatives to antibiotics.

Assessing the synergistic potential of bacteriophage endolysins and antimicrobial peptides for eradicating bacterial biofilms.

Phage-encoded endolysins have emerged as a potential substitute to conventional antibiotics due to their exceptional benefits including host specificity, rapid host killing, least risk of resistance. In addition to their antibacterial potency and biofilm eradication properties, endolysins are reported to exhibit synergism with other antimicrobial agents. In this study, the synergistic potency of endolysins was dissected with antimicrobial peptides to enhance their therapeutic effectiveness. Recombinantly expressed and purified bacteriophage endolysin [T7 endolysin (T7L); and T4 endolysin (T4L)] proteins have been used to evaluate the broad-spectrum antibacterial efficacy using different bacterial strains. Antibacterial/biofilm eradication studies were performed in combination with different antimicrobial peptides (AMPs) such as colistin, nisin, and polymyxin B (PMB) to assess the endolysin's antimicrobial efficacy and their synergy with AMPs. In combination with T7L, polymyxin B and colistin effectively eradicated the biofilm of Pseudomonas aeruginosa and exhibited a synergistic effect. Further, a combination of T4L and nisin displayed a synergistic effect against Staphylococcus aureus biofilms. In summary, the obtained results endorse the theme of combinational therapy consisting of endolysins and AMPs as an effective remedy against the drug-resistant bacterial biofilms that are a serious concern in healthcare settings.

Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes.

The molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, and Okazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis. We show that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis. Fluorescence imaging reveals polymerases remaining bound to the lagging strand behind the replication fork, consistent with Okazaki-fragment synthesis behind and independent of the replication complex. Individual replisomes display both looping and pausing during priming, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation.

Increasing CRISPR/Cas9-mediated gene editing efficiency in T7 phage by reducing the escape rate based on insight into the survival mechanism.

Bacteriophages have been used across various fields, and the utilization of CRISPR/Cas-based genome editing technology can accelerate the research and applications of bacteriophages. However, some bacteriophages can escape from the cleavage of Cas protein, such as Cas9, and decrease the efficiency of genome editing. This study focuses on the bacteriophage T7, which is widely utilized but whose mechanism of evading the cleavage of CRISPR/Cas9 has not been elucidated. First, we test the escape rates of T7 phage at different cleavage sites, ranging from 10 -2 to 10 -5. The sequencing results show that DNA point mutations and microhomology-mediated end joining (MMEJ) at the target sites are the main causes. Next, we indicate the existence of the hotspot DNA region of MMEJ and successfully reduce MMEJ events by designing targeted sites that bypass the hotspot DNA region. Moreover, we also knock out the ATP-dependent DNA ligase 1. 3 gene, which may be involved in the MMEJ event, and the frequency of MMEJ at 4. 3 is reduced from 83% to 18%. Finally, the genome editing efficiency in T7 Δ 1. 3 increases from 20% to 100%. This study reveals the mechanism of T7 phage evasion from the cleavage of CRISPR/Cas9 and demonstrates that the special design of editing sites or the deletion of key gene 1. 3 can reduce MMEJ events and enhance gene editing efficiency. These findings will contribute to advancing CRISPR/Cas-based tools for efficient genome editing in phages and provide a theoretical foundation for the broader application of phages.