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Viruses Jun 2015The T4 bacteriophage encodes eight proteins, which are sufficient to carry out coordinated leading and lagging strand DNA synthesis. These purified proteins have been... (Review)
Review
The T4 bacteriophage encodes eight proteins, which are sufficient to carry out coordinated leading and lagging strand DNA synthesis. These purified proteins have been used to reconstitute DNA synthesis in vitro and are a well-characterized model system. Recent work on the T4 replisome has yielded more detailed insight into the dynamics and coordination of proteins at the replication fork. Since the leading and lagging strands are synthesized in opposite directions, coordination of DNA synthesis as well as priming and unwinding is accomplished by several protein complexes. These protein complexes serve to link catalytic activities and physically tether proteins to the replication fork. Essential to both leading and lagging strand synthesis is the formation of a holoenzyme complex composed of the polymerase and a processivity clamp. The two holoenzymes form a dimer allowing the lagging strand polymerase to be retained within the replisome after completion of each Okazaki fragment. The helicase and primase also form a complex known as the primosome, which unwinds the duplex DNA while also synthesizing primers on the lagging strand. Future studies will likely focus on defining the orientations and architecture of protein complexes at the replication fork.
Topics: Bacteriophage T4; DNA Replication; DNA, Viral; Multienzyme Complexes; Viral Proteins
PubMed: 26102578
DOI: 10.3390/v7062766 -
Virology Journal Dec 2010Homologous recombination (HR), a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining... (Review)
Review
Homologous recombination (HR), a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining the genetic diversity and genome stability of species. Bacteriophage T4 is one of the classic systems for studies of homologous recombination. T4 uses HR for high-frequency genetic exchanges, for homology-directed DNA repair (HDR) processes including DNA double-strand break repair, and for the initiation of DNA replication (RDR). T4 recombination proteins are expressed at high levels during T4 infection in E. coli, and share strong sequence, structural, and/or functional conservation with their counterparts in cellular organisms. Biochemical studies of T4 recombination have provided key insights on DNA strand exchange mechanisms, on the structure and function of recombination proteins, and on the coordination of recombination and DNA synthesis activities during RDR and HDR. Recent years have seen the development of detailed biochemical models for the assembly and dynamics of presynaptic filaments in the T4 recombination system, for the atomic structure of T4 UvsX recombinase, and for the roles of DNA helicases in T4 recombination. The goal of this chapter is to review these recent advances and their implications for HR and HDR mechanisms in all organisms.
Topics: Bacterial Proteins; Bacteriophage T4; DNA Repair; DNA Replication; DNA, Viral; Escherichia coli; Models, Biological; Recombination, Genetic; Viral Proteins
PubMed: 21129202
DOI: 10.1186/1743-422X-7-357 -
Advances in Virus Research 2012The bacteriophage T4 head is an elongated icosahedron packed with 172 kb of linear double-stranded DNA and numerous proteins. The capsid is built from three essential... (Review)
Review
The bacteriophage T4 head is an elongated icosahedron packed with 172 kb of linear double-stranded DNA and numerous proteins. The capsid is built from three essential proteins: gp23*, which forms the hexagonal capsid lattice; gp24*, which forms pentamers at 11 of the 12 vertices; and gp20, which forms the unique dodecameric portal vertex through which DNA enters during packaging and exits during infection. Intensive work over more than half a century has led to a deep understanding of the phage T4 head. The atomic structure of gp24 has been determined. A structural model built for gp23 using its similarity to gp24 showed that the phage T4 major capsid protein has the same fold as numerous other icosahedral bacteriophages. However, phage T4 displays an unusual membrane and portal initiated assembly of a shape determining self-sufficient scaffolding core. Folding of gp23 requires the assistance of two chaperones, the Escherichia coli chaperone GroEL acting with the phage-coded gp23-specific cochaperone, gp31. The capsid also contains two nonessential outer capsid proteins, Hoc and Soc, which decorate the capsid surface. Through binding to adjacent gp23 subunits, Soc reinforces the capsid structure. Hoc and Soc have been used extensively in bipartite peptide display libraries and to display pathogen antigens, including those from human immunodeficiency virus (HIV), Neisseria meningitides, Bacillus anthracis, and foot and mouth disease virus. The structure of Ip1*, one of a number of multiple (>100) copy proteins packed and injected with DNA from the full head, shows it to be an inhibitor of one specific restriction endonuclease specifically targeting glycosylated hydroxymethyl cytosine DNA. Extensive mutagenesis, combined with atomic structures of the DNA packaging/terminase proteins gp16 and gp17, elucidated the ATPase and nuclease functional motifs involved in DNA translocation and headful DNA cutting. The cryoelectron microscopy structure of the T4 packaging machine showed a pentameric motor assembled with gp17 subunits on the portal vertex. Single molecule optical tweezers and fluorescence studies showed that the T4 motor packages DNA at the highest rate known and can package multiple segments. Förster resonance energy transfer-fluorescence correlation spectroscopy studies indicate that DNA gets compressed in the stalled motor and that the terminase-to-portal distance changes during translocation. Current evidence suggests a linear two-component (large terminase plus portal) translocation motor in which electrostatic forces generated by ATP hydrolysis drive DNA translocation by alternating the motor between tensed and relaxed states.
Topics: Adenosine Triphosphatases; Bacteriophage T4; Capsid; Capsid Proteins; DNA Packaging; DNA, Viral; Endodeoxyribonucleases; Escherichia coli; Nucleic Acid Conformation; Protein Conformation; Protein Folding; Virion; Virus Assembly
PubMed: 22420853
DOI: 10.1016/B978-0-12-394621-8.00018-2 -
Virology Journal Dec 2010Over 50 years of biological research with bacteriophage T4 includes notable discoveries in post-transcriptional control, including the genetic code, mRNA, and tRNA; the... (Review)
Review
Over 50 years of biological research with bacteriophage T4 includes notable discoveries in post-transcriptional control, including the genetic code, mRNA, and tRNA; the very foundations of molecular biology. In this review we compile the past 10-15 year literature on RNA-protein interactions with T4 and some of its related phages, with particular focus on advances in mRNA decay and processing, and on translational repression. Binding of T4 proteins RegB, RegA, gp32 and gp43 to their cognate target RNAs has been characterized. For several of these, further study is needed for an atomic-level perspective, where resolved structures of RNA-protein complexes are awaiting investigation. Other features of post-transcriptional control are also summarized. These include: RNA structure at translation initiation regions that either inhibit or promote translation initiation; programmed translational bypassing, where T4 orchestrates ribosome bypass of a 50 nucleotide mRNA sequence; phage exclusion systems that involve T4-mediated activation of a latent endoribonuclease (PrrC) and cofactor-assisted activation of EF-Tu proteolysis (Gol-Lit); and potentially important findings on ADP-ribosylation (by Alt and Mod enzymes) of ribosome-associated proteins that might broadly impact protein synthesis in the infected cell. Many of these problems can continue to be addressed with T4, whereas the growing database of T4-related phage genome sequences provides new resources and potentially new phage-host systems to extend the work into a broader biological, evolutionary context.
Topics: Bacteriophage T4; Gene Expression Regulation; Protein Biosynthesis; RNA Processing, Post-Transcriptional; RNA Stability; RNA, Messenger
PubMed: 21129205
DOI: 10.1186/1743-422X-7-360 -
Journal of Molecular Biology Sep 2019The parasitic life cycle of viruses involves the obligatory subversion of the host's macromolecular processes for efficient viral progeny production. Viruses that infect... (Review)
Review
The parasitic life cycle of viruses involves the obligatory subversion of the host's macromolecular processes for efficient viral progeny production. Viruses that infect bacteria, bacteriophages (phages), are no exception and have evolved sophisticated ways to control essential biosynthetic machineries of their bacterial prey to benefit phage development. The xenogeneic regulation of bacterial cell function is a poorly understood area of bacteriology. The activity of the bacterial transcription machinery, the RNA polymerase (RNAP), is often regulated by a variety of mechanisms involving small phage-encoded proteins. In this review, we provide a brief overview of known phage proteins that interact with the bacterial RNAP and compare how two prototypical phages of Escherichia coli, T4 and T7, use small proteins to "puppeteer" the bacterial RNAP to ensure a successful infection.
Topics: Bacterial Proteins; Bacteriophage T4; Bacteriophage T7; DNA-Directed RNA Polymerases; Escherichia coli; Gene Expression Regulation, Bacterial; Microbial Interactions; Transcription, Genetic; Viral Proteins
PubMed: 30776429
DOI: 10.1016/j.jmb.2019.02.008 -
Molecular Microbiology Apr 2018Contractile tail bacteriophages, or myobacteriophages, use a sophisticated biomolecular structure to inject their genome into the bacterial host cell. This structure... (Review)
Review
Contractile tail bacteriophages, or myobacteriophages, use a sophisticated biomolecular structure to inject their genome into the bacterial host cell. This structure consists of a contractile sheath enveloping a rigid tube that is sharpened by a spike-shaped protein complex at its tip. The spike complex forms the centerpiece of a baseplate complex that terminates the sheath and the tube. The baseplate anchors the tail to the target cell membrane with the help of fibrous proteins emanating from it and triggers contraction of the sheath. The contracting sheath drives the tube with its spiky tip through the target cell membrane. Subsequently, the bacteriophage genome is injected through the tube. The structural transformation of the bacteriophage T4 baseplate upon binding to the host cell has been recently described in near-atomic detail. In this review we discuss structural elements and features of this mechanism that are likely to be conserved in all contractile injection systems (systems evolutionary and structurally related to contractile bacteriophage tails). These include the type VI secretion system (T6SS), which is used by bacteria to transfer effectors into other bacteria and into eukaryotic cells, and tailocins, a large family of contractile bacteriophage tail-like compounds that includes the P. aeruginosa R-type pyocins.
Topics: Bacteriophage T4; Biological Evolution; Cell Membrane; Genome, Viral; Gram-Negative Bacteria; Pyocins; Type VI Secretion Systems; Viral Tail Proteins; X-Ray Diffraction
PubMed: 29405518
DOI: 10.1111/mmi.13921 -
Biochemistry. Biokhimiia Nov 2004In studying bacteriophage T4--one of the basic models of molecular biology for several decades--there has come a Renaissance, and this virus is now actively used as... (Review)
Review
In studying bacteriophage T4--one of the basic models of molecular biology for several decades--there has come a Renaissance, and this virus is now actively used as object of structural biology. The structures of six proteins of the phage particle have recently been determined at atomic resolution by X-ray crystallography. Three-dimensional reconstruction of the infection device--one of the most complex multiprotein components--has been developed on the basis of cryo-electron microscopy images. The further study of bacteriophage T4 structure will allow a better understanding of the regulation of protein folding, assembly of biological structures, and also mechanisms of functioning of the complex biological molecular machines.
Topics: Animals; Bacteriophage T4; Protein Conformation; Viral Proteins; Virus Assembly
PubMed: 15627372
DOI: 10.1007/s10541-005-0064-9 -
Virology May 2015DNA packaging into empty viral procapsids by ATP-driven motor proteins applies widely among viruses. Recent fluorescence studies of phage T4 reveal: 1) the small... (Review)
Review
DNA packaging into empty viral procapsids by ATP-driven motor proteins applies widely among viruses. Recent fluorescence studies of phage T4 reveal: 1) the small terminase subunit (TerS) synapses pac homologs by a twin ring mechanism to gauge DNA maturation and allow packaging by the large terminase subunit (TerL); 2) translocation of linear DNA is efficient by TerL acting alone; expansion of the procapsid is controlled by the portal-terminase assembly; 3) both ends of the packaged DNA are held at the portal, showing a loop of DNA is packaged; 4) transient spring-like compression of B form to A form-like DNA accompanies translocation; 5) the C-terminal domain of TerL is docked to the portal and moves toward it when stalled; 6) a portal bound resolvase can release stalled Y-DNA compression and allow translocation in vitro; and 7) ATP powered translocation on A form dsDNA is supported by recent hexameric helicase studies.
Topics: Bacteriophage T4; DNA Packaging; DNA, Viral; Models, Biological; Viral Proteins
PubMed: 25728298
DOI: 10.1016/j.virol.2015.01.015 -
Virology Journal Oct 2010This article reviews the current state of understanding of the regulated transcription of the bacteriophage T4 late genes, with a focus on the underlying biochemical... (Review)
Review
This article reviews the current state of understanding of the regulated transcription of the bacteriophage T4 late genes, with a focus on the underlying biochemical mechanisms, which turn out to be unique to the T4-related family of phages or significantly different from other bacterial systems. The activator of T4 late transcription is the gene 45 protein (gp45), the sliding clamp of the T4 replisome. Gp45 becomes topologically linked to DNA through the action of its clamp-loader, but it is not site-specifically DNA-bound, as other transcriptional activators are. Gp45 facilitates RNA polymerase recruitment to late promoters by interacting with two phage-encoded polymerase subunits: gp33, the co-activator of T4 late transcription; and gp55, the T4 late promoter recognition protein. The emphasis of this account is on the sites and mechanisms of actions of these three proteins, and on their roles in the formation of transcription-ready open T4 late promoter complexes.
Topics: Bacteriophage T4; DNA, Viral; Gene Expression Regulation, Viral; Genes, Viral; Promoter Regions, Genetic; Protein Binding; Transcription, Genetic; Viral Proteins
PubMed: 21029432
DOI: 10.1186/1743-422X-7-288 -
Nucleic Acids Research Sep 2019Bacteriophage T4 middle promoters are activated through a process called σ appropriation, which requires the concerted effort of two T4-encoded transcription factors:...
Bacteriophage T4 middle promoters are activated through a process called σ appropriation, which requires the concerted effort of two T4-encoded transcription factors: AsiA and MotA. Despite extensive biochemical and genetic analyses, puzzle remains, in part, because of a lack of precise structural information for σ appropriation complex. Here, we report a single-particle cryo-electron microscopy (cryo-EM) structure of an intact σ appropriation complex, comprising AsiA, MotA, Escherichia coli RNA polymerase (RNAP), σ70 and a T4 middle promoter. As expected, AsiA binds to and remodels σ region 4 to prevent its contact with host promoters. Unexpectedly, AsiA undergoes a large conformational change, takes over the job of σ region 4 and provides an anchor point for the upstream double-stranded DNA. Because σ region 4 is conserved among bacteria, other transcription factors may use the same strategy to alter the landscape of transcription immediately. Together, the structure provides a foundation for understanding σ appropriation and transcription activation.
Topics: Bacteriophage T4; DNA; DNA-Binding Proteins; DNA-Directed RNA Polymerases; Escherichia coli; Multiprotein Complexes; Promoter Regions, Genetic; Protein Conformation; Transcription Factors; Viral Proteins
PubMed: 31392983
DOI: 10.1093/nar/gkz682