Identification of the ssDNA-binding protein of bacteriophage T5: Implications for T5 replication.
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ABSTRACT:
SUBMITTER: Werten S
PROVIDER: S-EPMC3897522 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The Escherichia coli bacteriophage T5 has three temporal classes of genes (pre-early, early, and late). All three classes are transcribed by host RNA polymerase (RNAP) containing the σ70 promoter specificity subunit. Molecular mechanisms responsible for the switching of viral transcription from one class to another remain unknown. Here, we find the product of T5 gene 026 (gpT5.026) in RNAP preparations purified from T5-infected cells and demonstrate in vitro its tight binding to E. coli RNAP. While proteins homologous to gpT5.026 are encoded by all T5-related phages, no similarities to proteins with known functions can be detected. GpT5.026 binds to two regions of the RNAP β subunit and moderately inhibits RNAP interaction with the discriminator region of σ70-dependent promoters. A T5 mutant with disrupted gene 026 is viable, but the host cell lysis phase is prolongated and fewer virus particles are produced. During the mutant phage infection, the number of early transcripts increases, whereas the number of late transcripts decreases. We propose that gpT5.026 is part of the regulatory cascade that orchestrates a switch from early to late bacteriophage T5 transcription.
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Project description:Helicases catalyze the unwinding of double-stranded nucleic acids where structure and phosphate backbone contacts, rather than nucleobase sequence, usually determines substrate specificity. We have expressed and purified a putative helicase encoded by the D10 gene of bacteriophage T5. Here we report that this hitherto uncharacterized protein possesses branch migration and DNA unwinding activity. The initiation of substrate unwinding showed some sequence dependency, while DNA binding and DNA-dependent ATPase activity did not. DNA footprinting and purine-base interference assays demonstrated that D10 engages these substrates with a defined polarity that may be established by protein-nucleobase contacts. Bioinformatic analysis of the nucleotide databases revealed genes predicted to encode proteins related to D10 in archaebacteria, bacteriophages and in viruses known to infect a range of eukaryotic organisms.
| S-EPMC5180179 | biostudies-literature
Project description:Binding of bacteriophage T5 to its receptor, the Escherichia coli FhuA protein, is mediated by tail protein pb5. In this article we confirm that pb5 is encoded by the T5 oad gene and describe the isolation, expression, and sequencing of this gene. In order to locate oad precisely, we analyzed recombinants between BF23, a T5-related phage with a different host range, and plasmid clones containing segments of the T5 chromosome. This analysis also showed that oad has little or no homology with hrs, the analogous BF23 gene. We were able to overproduce a protein that comigrates with pb5 after fusing a 2-kb segment containing oad to a phage T7 promoter. This segment contains an open reading frame that can encode a protein of the appropriate size. Its deduced amino acid sequence does not closely resemble that of any other protein in the database. The sequence upstream of the open reading frame shows typical characteristics of a promoter region with two overlapping, divergently orientated promoters.
| S-EPMC207253 | biostudies-other
Project description:Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex. DNA primer/template (p/t) constructs are used as models to determine the effects of ssDNA lattice length, gp32 concentration, salt concentration, binding cooperativity and binding polarity at p/t junctions. Hidden Markov models (HMMs) and transition density plots (TDPs) are used to characterize the dynamics of the multi-step assembly pathway of gp32 at p/t junctions of differing polarity, and show that isolated gp32 molecules bind to their ssDNA targets weakly and dissociate quickly, while cooperatively bound dimeric or trimeric clusters of gp32 bind much more tightly, can 'slide' on ssDNA sequences, and exhibit binding dynamics that depend on p/t junction polarities. The potential relationships of these binding dynamics to interactions with other components of the T4 DNA replication complex are discussed.
| S-EPMC5159549 | biostudies-literature
Project description:Bacillus subtilis phage ?29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5' ends of the DNA. This protein, called terminal protein (TP), is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB) that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP) that preferentially binds to the origins of replication at the ?29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for ?29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in ?29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the ?29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3'-5' exonucleolysis and pyrophosphorolysis. The resolution of the ?29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made ?29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of ?29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding and localization of the TP at the bacterial nucleoid, where viral DNA replication takes place. The biochemical properties of the ?29 DBP and SSB and their function in the initiation and elongation of ?29 DNA replication, respectively, will be described.
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Project description: Not available
| EMPIAR-11319 | biostudies-other
Project description:Bacteriophage capsids constitute icosahedral shells of exceptional stability that protect the viral genome. Many capsids display on their surface decoration proteins whose structure and function remain largely unknown. The decoration protein pb10 of phage T5 binds at the centre of the 120 hexamers formed by the major capsid protein. Here we determined the 3D structure of pb10 and investigated its capsid-binding properties using NMR, SAXS, cryoEM and SPR. Pb10 consists of an α-helical capsid-binding domain and an Ig-like domain exposed to the solvent. It binds to the T5 capsid with a remarkably high affinity and its binding kinetics is characterized by a very slow dissociation rate. We propose that the conformational exchange events observed in the capsid-binding domain enable rearrangements upon binding that contribute to the quasi-irreversibility of the pb10-capsid interaction. Moreover we show that pb10 binding is a highly cooperative process, which favours immediate rebinding of newly dissociated pb10 to the 120 hexamers of the capsid protein. In extreme conditions, pb10 protects the phage from releasing its genome. We conclude that pb10 may function to reinforce the capsid thus favouring phage survival in harsh environments.
| S-EPMC5292684 | biostudies-literature
Project description:Bacteriophage T5 represents a large family of lytic Siphoviridae infecting Gram-negative bacteria. The low-resolution structure of T5 showed the T=13 geometry of the capsid and the unusual trimeric organization of the tail tube, and the assembly pathway of the capsid was established. Although major structural proteins of T5 have been identified in these studies, most of the genes encoding the morphogenesis proteins remained to be identified. Here, we combine a proteomic analysis of T5 particles with a bioinformatic study and electron microscopic immunolocalization to assign function to the genes encoding the structural proteins, the packaging proteins, and other nonstructural components required for T5 assembly. A head maturation protease that likely accounts for the cleavage of the different capsid proteins is identified. Two other proteins involved in capsid maturation add originality to the T5 capsid assembly mechanism: the single head-to-tail joining protein, which closes the T5 capsid after DNA packaging, and the nicking endonuclease responsible for the single-strand interruptions in the T5 genome. We localize most of the tail proteins that were hitherto uncharacterized and provide a detailed description of the tail tip composition. Our findings highlight novel variations of viral assembly strategies and of virion particle architecture. They further recommend T5 for exploring phage structure and assembly and for deciphering conformational rearrangements that accompany DNA transfer from the capsid to the host cytoplasm.
| S-EPMC3911642 | biostudies-literature
Project description:Human Replication Protein A (hRPA) is a multidomain protein that interacts with ssDNA intermediates to provide the latter much-needed stability during DNA metabolism and maintain genomic integrity. Although the ssDNA organization with hRPA was studied recently through experimental means, characterizing the underlying mechanism at the atomic level remains challenging because of the dynamic domain architecture of hRPA and poorly understood heterogeneity of ssDNA-protein interactions. Here, we used a computational framework, precisely tailored to capture protein-ssDNA interactions, and investigated the binding of hRPA with a 60 nt ssDNA. Two distinct binding mechanisms are realized based on the hRPA domain flexibility. For a rigid domain architecture of hRPA, ssDNA binds sequentially with hRPA domains, resulting in slow association kinetics. The binding pathway involves the formation of stable and distinct intermediate states. On contrary, for a flexible domain architecture of hRPA, ssDNA binds synergistically to the A and B domains followed by the rest of hRPA. The domain dynamics in hRPA alleviates the free energy cost of domain orientation necessary for specific binding with ssDNA, leading to fast association kinetics along a downhill binding free energy landscape. An ensemble of free energetically degenerate intermediate states is encountered that makes it arduous to characterize them structurally. An excellent match between our results with the available experimental observations provides new insights into the rich dynamics of hRPA binding to ssDNA and in general paves the way to investigate intricate details of ssDNA-protein interactions, crucial for cellular functioning.
| S-EPMC9851514 | biostudies-literature
Project description:Coliphage T5 injects its DNA in 2 steps: the first step transfer (FST) region 7.9% is injected and its genes are expressed and only then does the remainder (second step transfer, SST) of its DNA enter the cell. In the FST region, only 2 essential genes (A1 and A2) have been identified and a third (dmp) non-essential gene codes for a deoxyribonucleotide 5' monophosphatase. Thirteen additional putative ORFs are present in the FST region. Numerous properties have been attributed to FST region, including SST, host DNA degradation, inhibition of host RNA and protein synthesis, restriction insensitivity and protection of T5 DNA. These effects do not occur following infection with an A1 mutant. The A2 gene seems only to be involved in SST transfer. This is puzzling since there are more seemingly unrelated effects than there are essential genes to accomplish them and it is possible that some important genes were not identified. This review attempts to analyze these problems that were first identified in the 1970-80 s. In particular, an attempt is made to determine which potential ORFs are conserved in evolution (and thus likely to be important); by comparing T5 to 10 newly isolated and completely sequenced T5-like phages. A similar approach is used to identify conserved repeats, inverted repeats and palindromes that occur in all T5-like phages in the region containing the injection stop signal (iss) and the terminase substrate. Finally, an attempt is made to re-analyze the mechanism whereby T5 protects itself from the enzymes that degrade host DNA, from the RecBCD nuclease and from restriction enzymes. For all of these FST effects new hypotheses and possible new genetic and biochemical approaches are envisaged.
| S-EPMC4743489 | biostudies-literature