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1.
JoséL. Carrascosa Eladio Viñuela Narciso García Antonio Santisteban 《Journal of molecular biology》1982,154(2):311-324
The head-to-tail connecting region of bacteriophage φ29 has been studied by isolating neck-tail complexes from disrupted phage. These complexes can be isolated with appendages (from wild-type phage) or without appendages (from phage mutant sus12). Treatment of the neck-tail complex without appendages with urea or guanidinium hydrochloride releases the tail protein (p9) from the neck complex (proteins p10 and p11). Electron micrographs of φ29 necks show that they are composed of two collars and a thin axial tube. There is an internal hole along the longitudinal axis, from the upper collar to the thin tube.Image-processing analysis of electron micrographs of two-dimensional crystals of necks shows that the neck of phage φ29 consists of 12 external units and an internal area of apparent 6-fold symmetry, with a hole in the centre. 相似文献
2.
Three-dimensional reconstruction of the connector of bacteriophage phi 29 at 1.8 nm resolution 总被引:8,自引:0,他引:8
J M Carazo L E Donate L Herranz J P Secilla J L Carrascosa 《Journal of molecular biology》1986,192(4):853-867
The three-dimensional reconstruction of the connector of bacteriophage phi 29 has been obtained from tilt series of negatively stained tetragonal ordered aggregates under low-dose conditions and up to a resolution of (1/1.8) nm-1. These connectors are built up as dodecamers of only one structural polypeptide (p10). Two connectors form the crystal unit cell, each one facing in the opposite direction with respect to the plane of the crystal and partially overlapping. The main features of the two connectors that build the unit cell were essentially the same, although they were negatively stained in slightly different ways, probably due to their situations with respect to the carbon-coated support grid. The main features of the phi 29 connector structure revealed by this three-dimensional reconstruction are: the existence of two clearly defined domains, one with a diameter of around 14 nm and the other narrower (diameter approximately equal to 7.5 nm); an inner hole running all along the structure (around 7 to 8 nm in height) with a cylindrical profile and an average diameter of 4 nm; a general 6-fold symmetry along the whole structure and a 12-fold one in the wider domain; a clockwise twist of the more contrasted regions of both domains from the narrower towards the wider domain (the direction of DNA encapsidation). These features are compatible with an active role for the connector in the process of DNA packaging. 相似文献
3.
Alejandro Serna-Rico Margarita Salas Wilfried J J Meijer 《The Journal of biological chemistry》2002,277(8):6733-6742
The functional role of the phi 29-encoded integral membrane protein p16.7 in phage DNA replication was studied using a soluble variant, p16.7A, lacking the N-terminal membrane-spanning domain. Because of the protein-primed mechanism of DNA replication, the bacteriophage phi 29 replication intermediates contain long stretches of single-stranded DNA (ssDNA). Protein p16.7A was found to be an ssDNA-binding protein. In addition, by direct and functional analysis we show that protein p16.7A binds to the stretches of ssDNA of the phi 29 DNA replication intermediates. Properties of protein p16.7A were compared with those of the phi 29-encoded single-stranded DNA-binding protein p5. The results obtained show that both proteins have different, non-overlapping functions. The likely role of p16.7 in attaching phi 29 DNA replication intermediates to the membrane of the infected cell is discussed. Homologues of gene 16.7 are present in phi 29-related phages, suggesting that the proposed role of p16.7 is conserved in this family of phages. 相似文献
4.
Order of assembly of the lower collar and the tail proteins of Bacillus subtilis bacteriophage phi 29.
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Extracts obtained after restrictive infection of Bacillus subtilis with mutants in cistron 11 of bacteriophage phi 29 are complemented in vitro by extract donors of the lower collar protein (p11). Purified 11- heads, containing the major capsid protein (p8), the fiber protein (p8.5), the upper collar protein (p10), and the virus DNA, can be also complemented in vitro to produce infective virus. This result suggests that 11- heads are intermediates in phage phi 29 morphogenesis. The order of assembly of the lower collar protein p11 and the tail protein p9 was determined in vitro in two complementation steps. The results obtained indicate that the lower collar protein is assembled before the tail protein. 相似文献
5.
J M Valpuesta J J Fernández J M Carazo J L Carrascosa 《Structure (London, England : 1993)》1999,7(3):289-296
BACKGROUND: Head-tail connectors are viral substructures that are very important in the viral morphogenetic cycle, having roles in the formation of the precursor capsid (prohead), DNA packaging, tail binding to the mature head and in the infection process. Structural information on the connector would, therefore, help us to understand how this structure is related to a multiplicity of functions. RESULTS: Recombinant bacteriophage phi29 connectors have been crystallized in two-dimensional aggregates. An average projection image and a three-dimensional map have been obtained at 8 A and 10 A resolution, respectively, from untilted and tilted images of vitrified specimens of the two-dimensional crystals. The average projection image reveals a central mass surrounding a channel with 12 appendages protruding from the central mass. The three-dimensional map reveals a wide domain surrounded by 12 appendages that interact with the prohead vertex, and a narrow domain that interacts with the bacteriophage tail. At the junction of the two domains, 12 smaller appendages are visualized. A channel runs along the axis of the connector structure and is sufficiently wide to allow a double-stranded DNA molecule to pass through. CONCLUSIONS: The propeller-like structure of the phi29 connector strengthens the notion of the connector rotating during DNA packaging. The groove formed by the two lanes of large and small appendages may act as a rail to prevent the liberation of the connector from the prohead vertex during rotation. 相似文献
6.
Characterization of the bacteriophage phi29-encoded protein p16.7: a membrane protein involved in phage DNA replication 总被引:1,自引:0,他引:1
An early expressed operon, located at the right end of the linear bacteriophage phi29 genome, contains open reading frame (ORF)16.7, whose deduced protein sequence of 130 amino acids is conserved in phi29-related phages. Here, we show that this ORF actually encodes a protein, p16.7, which is abundantly and early expressed after infection. p16.7 is a membrane protein, and the N-terminally located transmembrane-spanning domain is required for its membrane localization. The variant p16.7A, in which the N-terminal membrane anchor was replaced by a histidine-tag, was purified and characterized. Purified p16.7A was shown to form dimers in solution. To study the in vivo role of p16.7, a phi29 mutant containing a suppressible mutation in gene 16.7 was constructed. In vivo phage DNA replication was affected in the absence of p16.7, especially at early infection times. Based on the results, the putative role of p16.7 in in vivo phi29 DNA replication is discussed. 相似文献
7.
Bacteriophage phi29 is one of the smallest and simplest known dsDNA phages, making it amenable to structural investigations. The three-dimensional structure of a fiberless, isometric variant has been determined to 7.9 A resolution by cryo-electron microscopy (cryo-EM), allowing the identification of alpha helices and beta sheets. Their arrangement indicates that the folds of the phi29 and bacteriophage HK97 capsid proteins are similar except for an additional immunoglobulin-like domain of the phi29 protein. An atomic model that incorporates these two domains fits well into the cryo-EM density of the T = 3, fiberless isometric phi29 particle, and cryo-EM structures of fibered isometric and fiberless prolate prohead phi29 particles at resolutions of 8.7 A and 12.7 A, respectively. Thus, phi29 joins the growing number of phages that utilize the HK97 capsid structure, suggesting that this protein fold may be as prevalent in capsids of dsDNA phages as the jelly roll fold is in eukaryotic viruses. 相似文献
8.
Compartmentalization of phage phi29 DNA replication: interaction between the primer terminal protein and the membrane-associated protein p1
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The bacteriophage phi29 replication protein p1 (85 amino acids) is membrane associated in Bacillus subtilis-infected cells. The C-terminal 52 amino acid residues of p1 are sufficient for assembly into protofilament sheet structures. Using chemical cross-linking experiments, we demonstrate here that p1DeltaC43, a C-terminally truncated p1 protein that neither associates with membranes in vivo nor self-interacts in vitro, can interact with the primer terminal protein (TP) in vitro. Like protein p1, plasmid-encoded protein p1DeltaC43 reduces the rate of phi29 DNA replication in vivo in a dosage-dependent manner. We also show that truncated p1 proteins that retain the N-terminal 42 amino acids, when present in excess, interfere with the in vitro formation of the TP.dAMP initiation complex in a reaction that depends on the efficient formation of a primer TP-phi29 DNA polymerase heterodimer. This interference is suppressed by increasing the concentration of either primer TP or phi29 DNA polymerase. We propose a model for initiation of in vivo phi29 DNA replication in which the viral replisome attaches to a membrane-associated p1-based structure. 相似文献
9.
Agirrezabala X Martín-Benito J Valle M González JM Valencia A Valpuesta JM Carrascosa JL 《Journal of molecular biology》2005,347(5):895-902
The three-dimensional structure of the bacteriophage T7 head-to-tail connector has been obtained at 8A resolution using cryo-electron microscopy and single-particle analysis from purified recombinant connectors. The general morphology of the T7 connector is that of a 12-folded toroidal homopolymer with a channel that runs along the longitudinal axis of the particle. The structure of the T7 connector reveals many structural similarities with the connectors from other bacteriophages. Docking of the atomic structure of the varphi29 connector into the three-dimensional reconstruction of T7 connector reveals that the narrow, distal region of the two oligomers are almost identical. This region of the varphi29 connector has been suggested to be involved in DNA translocation, and is composed of an alpha-beta-alpha-beta-beta-alpha motif. A search for alpha-helices in the same region of the T7 three-dimensional map has located three alpha-helices in approximately the same position as those of the varphi29 connector. A comparison of the predicted secondary structure of several bacteriophage connectors, including among others T7, varphi29, P22 and SPP1, reveals that, despite the lack of sequence homology, they seem to contain the same alpha-beta-alpha-beta-beta-alpha motif as that present in the varphi29 connector. These results allow us to suggest a common architecture related to a basic component of the DNA translocating machinery for several viruses. 相似文献
10.
Assembly of phage phi 29 genome with viral protein p6 into a compact complex. 总被引:4,自引:1,他引:3
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The formation of a multimeric nucleoprotein complex by the phage phi 29 dsDNA binding protein p6 at the phi 29 DNA replication origins, leads to activation of viral DNA replication. In the present study, we have analysed protein p6-DNA complexes formed in vitro along the 19.3 kb phi 29 genome by electron microscopy and micrococcal nuclease digestion, and estimated binding parameters. Under conditions that greatly favour protein-DNA interaction, the saturated phi 29 DNA-protein p6 complex appears as a rigid, rod-like, homogeneous structure. Complex formation was analysed also by a psoralen crosslinking procedure that did not disrupt complexes. The whole phi 29 genome appears, under saturating conditions, as an irregularly spaced array of complexes approximately 200-300 bp long; however, the size of these complexes varies from approximately 2 kb to 130 bp. The minimal size of the complexes, confirmed by micrococcal nuclease digestion, probably reflects a structural requirement for stability. The values obtained for the affinity constant (K(eff) approximately 10(5) M-1) and the cooperativity parameter (omega approximately 100) indicate that the complex is highly dynamic. These results, together with the high abundance of protein p6 in infected cells, lead us to propose that protein p6-DNA complexes could have, at least at some stages, during infection, a structural role in the organization of the phi 29 genome into a nucleoid-type, compact nucleoprotein complex. 相似文献
11.
Replication of phage phi 29 DNA in vitro: role of the viral protein p6 in initiation and elongation. 总被引:15,自引:6,他引:9
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The phi 29 protein p6 stimulates the formation of the protein p3-dAMP initiation complex when added to a minimal system containing the terminal protein p3, the phi 29 DNA polymerase p2 and phi 29 DNA-protein p3 complex, by decreasing about 5 fold the Km value for dATP. In addition, protein p6 stimulates elongation of the p3-dAMP initiation complex. Whereas the effect of protein p6 on initiation is similar with protein p3-containing fragments from the right or left phi 29 DNA ends, the stimulation of elongation is higher with the right than with the left phi 29 DNA terminal fragment, suggesting DNA sequence specificity. The stimulation by protein p6 of the initiation and elongation steps of phi 29 DNA replication does not require the presence of the parental protein p3 at the phi 29 DNA ends. No effect of protein p6 was obtained on the elongation of the template-primer poly(dT)-(dA) 12-18 by the phi 29 DNA polymerase. 相似文献
12.
Signals at the bacteriophage phi 29 DNA replication origins required for protein p6 binding and activity. 总被引:7,自引:2,他引:5
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Protein p6 of Bacillus subtilis phage phi 29 binds specifically to the ends of the viral DNA that contain the replication origins, giving rise to a nucleoprotein structure. DNA regions recognized by protein p6 have been mapped by deletion analysis and DNase I footprinting. Main protein p6-recognition signals have been located between nucleotides 62 and 125 at the right phi 29 DNA end and between nucleotides 46 and 68 at the left end. In addition, recognition signals are also present at other sites within 200-300 bp at each phi 29 DNA end. Protein p6 does not seem to recognize a specific sequence in the DNA, but rather a structural feature, which could be bendability. The formation of the protein p6-DNA nucleoprotein complex is likely to be the structural basis for the protein p6 activity in the initiation of replication. 相似文献
13.
Comparison of the physical properties and assembly pathways of the related bacteriophages T7, T3 and phi II 总被引:4,自引:0,他引:4
To understand constraints on the evolution of bacteriophage assembly, the structures, electrophoretic mobilities (mu) and assembly pathways of the related double-stranded DNA bacteriophages T7, T3 and phi II, have been compared. The characteristics of the following T7, T3 and phi II capsids in these assembly pathways have also been compared: (1) a DNA-free procapsid (capsid I) that packages DNA during assembly; (b) a DNA packaging-associated conversion product of capsid I (capsid II). The molecular weights of the T3 and phi II genomes were 25.2 X 10(6) and 25.9 (+/- 0.2) X 10(6) (26.44 X 10(6) for T7, as previously determined), as determined by agarose gel electrophoresis of intact genomes. The radii of T7, T3 and phi II bacteriophages were indistinguishable by sieving during agarose gel electrophoresis (+/- 4%) and measurement of the bacteriophage hydration (+/- 2%) (30.1 nm for T7, as previously determined). Assuming a T = 7 icosahedral lattice for the arrangement of the major capsid subunits (p10A) of T7, T3 and phi II best explains these data and data previously obtained for T7. At pH 7.4 and an ionic strength of 1.2, the solid-support-free mu values (mu 0 values) of T7, T3 and phi II bacteriophages, obtained by extrapolation of mu during agarose gel electrophoresis to an agarose concentration of 0 and correction for electro-osmosis, were -0.71, -0.91 and -1.17(X 10(-4) cm2V-1 s-1. The mu 0 values of T7, T3 and phi II capsids I were -1.51, -1.58 and -2.07(X 10(-4] cm2V-1 s-1. For the capsids II, these mu 0 values were -0.82, -1.07 and -1.37(X 10(-4] cm2V-1 s-1. The tails of all three bacteriophages were positively charged and the capsid envelopes (heads) were negatively charged. In all cases the procapsid had a negative mu 0 value larger in magnitude than the negative mu 0 value for bacteriophage or capsid II. A trypsin-sensitive region in capsid I-associated, but not capsid II-associated, T3 p10A was observed (previously observed for T7). The largest fragment of trypsinized capsid I-associated p10A had the same molecular weight in T7 and T3, although the T3 p10A is 18% more massive than the T7 p10A. It is suggested that the trypsin-resistant region of capsid I-associated p10A determines the radius of the bacteriophage capsid. 相似文献
14.
Structure of Bacillus subtilis bacteriophage phi 29 and the length of phi 29 deoxyribonucleic acid 总被引:47,自引:4,他引:43
Anderson, D. L. (University of Minnesota, Minneapolis), D. D. Hickman, and B. E. Reilly. Structure of Bacillus subtilis bacteriophage phi29 and the length of phi29 deoxyribonucleic acid. J. Bacteriol. 91:2081-2089. 1966-Bacillus subtilis bacteriophage phi29 were negatively stained with phosphotungstic acid. The head of phi29 has a hexagonal outline with a flattened base, and is about 315 A wide and 415 A in length. The virus has an intricate tail about 325 A in length. Twelve spindle-shaped appendages are attached to the lower of two collars which comprise the proximal portion of the tail. The distal 130 A of the tail axis has a diameter of about 60 A and is larger in diameter than the axis of the upper portion of the tail. Comparison of electron microscopic counts of phi29 with plaque-forming units indicated that about 50% of the microscopic entities were infective. Phenol-extracted phi29 deoxyribonucleic acid (DNA) molecules were prepared for electron microscopy by the cytochrome c film technique of Kleinschmidt et al. Measurement of contour lengths of DNA molecules from three preparations gave skewed distributions of lengths with observed modal class values ranging from 5.7 to 5.9 mu. Assuming that phi29 DNA is a double helix in the B form, the corresponding molecular weights would be 10.9 x 10(6) to 11.3 x 10(6) daltons. The largest DNA molecules would have a volume of 1.9 x 10(7) A(3) which is about 25% greater than the estimated 1.4 x 10(7) A(3) internal volume of the phage head. 相似文献
15.
Protein topography of Sulfolobus solfataricus ribosomes by cross-linking with 2-iminothiolane. Sso L12e, Sso L10e, and Sso L11e are neighbors 总被引:1,自引:0,他引:1
Large ribosomal subunits from Sulfolobus solfataricus were cross-linked with 2-iminothiolane in order to investigate the arrangement of proteins in the region containing the multicopy acidic protein Sso L12e, the protein homologous to Escherichia coli L7/L12. Proteins from cross-linked 50 S subunits were extracted and fractionated by chromatography on CM-cellulose. Fractions containing Sso L12e were analyzed by "diagonal" (two-dimensional reducing/nonreducing) dodecyl sulfate polyacrylamide gel electrophoresis. Sso L12e appeared in cross-linked homodimers and also in cross-linked complexes that contained Sso L10e, the protein equivalent to E. coli L10. In addition, Sso L12e was found in cross-links to L4, L6a, L26, and L29. N-terminal sequences obtained for L6a and L26 showed them to have significant homologies to E. coli proteins L11 and L23, respectively. The results indicate the presence in this archaebacterial ribosome of Sso L12e dimers and their location near Sso L10e and Sso L11e. The Sso L12e-L29 (Sso L23e) cross-link suggests proximity between components of the factor-binding and peptidyltransferase domains, since E. coli L23 is a protein affinity-labeled by puromycin. The (Sso L12e)4-Sso L10 pentameric complex, identified previously from studies in solution, appears to represent correctly the arrangement of these proteins in the ribosome. The occurrence in the archaebacterial ribosome of this unique structural element, similar to those shown previously in eubacteria and eukaryotes, reinforces the concept that the protein quaternary structure of the ribosomal factor-binding domain is highly conserved. 相似文献
16.
Sequencing of the phi 29 DNA region [open reading frames (ORFs) 12, 11 and 10] between genes 6 and 4 of the mutant ts5(219) showed that a G in the wild-type phage had been changed to an A in the mutant at position 218 of ORF 10 indicating that this ORF corresponds to gene 5. ORF 10 was cloned in plasmid pPLc28 under the control of the PL promoter of phage lambda and, after heat induction of the Escherichia coli cells carrying the recombinant plasmid pGM26, a 12-kDa protein was overproduced, accounting for about 5% of the de novo synthesized protein. Introduction of a nonsense mutation in ORF 10 indicated that the latter codes for the 12-kDa protein. The predicted secondary structure, the hydrophilicity values and the antigenic regions of protein p5 are discussed. 相似文献
17.
Polymerization of bacteriophage phi 29 replication protein p1 into protofilament sheets. 总被引:1,自引:0,他引:1
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Protein p1 (85 amino acids) of the Bacillus subtilis phage phi29 is a membrane-associated protein required for in vivo viral DNA replication. In the present study, we have constructed two fusion proteins, maltose-binding protein (MalE)-p1 and MalE-p1DeltaN33. By using both sedimentation assays and negative-stain electron microscopy analysis, we demonstrated that MalE-p1 molecules self-associated into long filamentous structures, which did not assemble further into larger arrays. These structures were constituted by a core of protein p1 surrounded by MalE subunits. After removal of the MalE component by cleavage with protease factor Xa, the resulting protein p1 filaments tended to associate, forming bundles. The MalE-p1DeltaN33 fusion protein, however, did not self-interact in solution. Nevertheless, after being separated from the MalE domain by factor Xa digestion, protein p1DeltaN33 assembled into long protofilaments that associated in a highly ordered, parallel array forming large two-dimensional sheets. These structures resemble eukaryotic tubulin and bacterial FtsZ polymers. In addition, we show that protein p1 influences the rate of in vivo phi29 DNA synthesis in a temperature-dependent manner. We propose that protein p1 is a component of a viral-encoded structure that associates with the bacterial membrane. This structure would provide an anchoring site for the viral DNA replication machinery. 相似文献
18.
Transformation of Rhodopseudomonas sphaeroides with deoxyribonucleic acid isolated from bacteriophage R phi 6P. 总被引:2,自引:2,他引:0
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The transformation of the photosynthetic bacterium Rhodopseudomonas sphaeroides with the circular genome of the penicillinase-encoding, temperate bacteriophage R phi 6P was demonstrated. The transformation was dependent on the infection of the recipient by another, apparently closely related, temperature bacteriophage, R phi 9. Optimum transformation occurred in the recipient cells already lysogenic for R phi 9 when superinfected with that bacteriophage at multiplicities of infection between 1 and 10 R phi 9 particles per recipient cell. 相似文献
19.
Temperate Bacteriophage Infectious for Asporogenic Variants of Bacillus pumilus 总被引:6,自引:2,他引:4
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Bacillus pumilus strain NRRL B-3275 is lysogenic for an inducible, nondefective temperate bacteriophage phi75. phi75 infects and lysogenizes several asporogenic mutants of B. pumilus strain NRS 576 but does not productively infect the spore(+) parent. phi75 DNA is a linear duplex with a mol wt of about 29 x 10(6) and a buoyant density of 1.701 g/cm(3). The location of the phi75 prophage attachment site on the chromosome of both host strains is adjacent to a lysine marker. The apparent order is phi75 att lys trp. 相似文献
20.
Structural analysis of staphylococcal bacteriophage phi 11 attachment sites. 总被引:7,自引:4,他引:3
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The lysogenization of bacteriophage phi 11 in Staphylococcus aureus occurs by site-specific recombination. The DNA segments containing the attachment sites on the host chromosome, the phage genome, and the two junctions created by insertion of the prophage were cloned, and the nucleotide sequences were determined. The attachment sites share a very short common sequence of 10 base pairs. 相似文献