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At least 163 records · Page 9Linked to original sources
PubMed2005

Construction and 3-D computer modeling of connector arrays with tetragonal to decagonal transition induced by pRNA of phi29 DNA-packaging motor.

The bottom-up assembly of patterned arrays is an exciting and important area in current nanotechnology. Arrays can be engineered to serve as components in chips for a virtually inexhaustible list of applications ranging from disease diagnosis to ultrahigh-density data storage. In attempting to achieve this goal, a number of methods to facilitate array design and production have been developed. Cloning and expression of the gene coding for the connector of the bacterial virus phi29 DNA-packaging motor, overproduction of the gene products, and the in vitro construction of large-scale carpet-like arrays composed of connector are described in this report. The stability of the arrays under various conditions, including varied pH, temperature and ionic strength, was tested. The addition of packaging RNA (pRNA) into the array caused a dramatic shift in array structure, and resulted in the conversion of tetragonal arrays into larger decagonal structures comprised of both protein and RNA. RNase digestion confirmed that the conformational shift was caused by pRNA, and that RNA was present in the decagons. As has been demonstrated in biomotors, conformational shift of motor components can generate force for motor motion. The conformational shift reported here can be utilized as a potential force-generating mechanism for the construction of nanomachines. Three-dimensional computer models of the constructed arrays were also produced using a variety of connector building blocks with or without the N- or C-terminal sequence, which is absent from the current published crystal structures. Both the connector array and the decagon are ideal candidates to be used as templates to build patterned suprastructures in nanotechnology.

Bacillus Phages↗
PubMed2006

Structural changes of bacteriophage phi29 upon DNA packaging and release.

Cryo-electron microscopy three-dimensional reconstructions have been made of mature and of emptied bacteriophage phi29 particles without making symmetry assumptions. Comparisons of these structures with each other and with the phi29 prohead indicate how conformational changes might initiate successive steps of assembly and infection. The 12 adsorption capable 'appendages' were found to have a structure homologous to the bacteriophage P22 tailspikes. Two of the appendages are extended radially outwards, away from the long axis of the virus, whereas the others are around and parallel to the phage axis. The appendage orientations are correlated with the symmetry-mismatched positions of the five-fold related head fibers, suggesting a mechanism for partial cell wall digestion upon rotation of the head about the tail when initiating infection. The narrow end of the head-tail connector is expanded in the mature virus. Gene product 3, bound to the 5' ends of the genome, appears to be positioned within the expanded connector, which may potentiate the release of DNA-packaging machine components, creating a binding site for attachment of the tail.

Bacillus Phages↗
PubMed1990

Bacteriophage T7 DNA packaging. I. Plasmids containing a T7 replication origin and the T7 concatemer junction are packaged into transducing particles during phage infection.

Bacteriophage T7 DNA is a linear duplex molecule with a 160 base-pair direct repeat (terminal redundancy) at its ends. During replication, large DNA concatemers are formed, which are multimers of the T7 genome linked head to tail through recombination at the terminal redundancy. We define the sequence that results from this recombination, a mature right end joined to the left end of T7 DNA, as the concatemer junction. To study the processing and packaging of T7 concatemers into phage particles, we have cloned the T7 concatemer junction into a plasmid vector. This plasmid is efficiently (at least 15 particles/infected cell) packaged into transducing particles during a T7 infection. These transducing particles can be separated from T7 phage by sedimentation to equilibrium in CsCl. The packaged plasmid DNA is a linear concatemer of about 40 x 10(3) base-pairs with ends at the expected T7 DNA sequences. Thus, the T7 concatemer junction sequence on the plasmid is recognized for processing and packaging by the phage system. We have identified a T7 DNA replication origin near the right end of the T7 genome that is necessary for efficient plasmid packaging. The origin, which is associated with a T7 RNA polymerase promoter, causes amplification of the plasmid DNA during T7 infection. The amplified plasmid DNA sediments very rapidly and contains large concatemers, which are expected to be good substrates for the packaging reaction. When cloned in pBR322, a sequence containing only the mature right end of T7 DNA is sufficient for efficient packaging. Since this sequence does not contain DNA to the right of the site where a mature T7 right end is formed, it was expected that right ends would not form on this DNA. In fact, with this plasmid the right end does not form at the normal T7 sequence but is instead formed within the vector. Apparently, the T7 packaging system can also recognize a site in pBR322 DNA to produce an end for packaging. This site is not recognized solely by a "headful" mechanism, since there can be considerable variation in the amount of DNA packaged (32 x 10(3) to 42 x 10(3) base-pairs). Furthermore, deletion of this region from the vector DNA prevents packaging of the plasmid. The end that is formed in vector DNA is somewhat heterogeneous. About one-third of the ends are at a unique site (nucleotide 1712 of pBR322), which is followed by the sequence 5'-ATCTGT-3'. This sequence is also found adjacent to the cut made in a T7 DNA concatemer to produce a normal T7 right end.

Base Sequence↗
PubMed1987

A novel in vitro DNA packaging system demonstrating a direct role for the bacteriophage lambda FI gene product.

A new in vitro bacteriophage lambda DNA packaging system is described in which all the proteins necessary for head morphogenesis are supplied by extracts of plasmid-transformed cells. This assay is used to demonstrate that the lambda FI gene product (gpFI) is necessary for maximal packaging efficiency when proheads and terminase are present in limiting amounts. A 100- to 200-fold decrease in packaging is seen when gpFI is omitted. gpFI is shown to act at and/or after the stage in packaging where proheads bind to the DNA:terminase complex.

Bacteriophage lambda↗
PubMed1994

A novel method for generating nested deletions using the in vitro bacteriophage T3 DNA packaging system.

To sequence a DNA segment inserted into a cosmid vector under the directed sequencing strategy, we established a simple and rapid method for generating nested deletions which uses the in vitro packaging system of bacteriophage T3 DNA. The principle is based on the previous finding that this system can translocate any linear double-stranded DNA up to 40 kb into the phage capsid in a time-dependent manner and the encapsulated DNA becomes DNase-resistant. For this purpose, we constructed a cosmid vector that carries two different antibiotic selection markers at both sides of the multiple cloning site, and after insertion of a DNA segment, the clone was linearized by lambda-terminase at the cos site. After the packaging reaction in vitro followed by DNase treatment, the encapsulated DNA was introduced into Escherichia coli cells to give clones with unidirectional deletions by differential antibiotic selection. Restriction and sequence analyses of deletion clones demonstrated that an ordered set of clones with nested deletions, ranging from less than 1 kb to 25 kb, was created from either the end of the DNA segment. Thus, nested deletion clones that cover the entire region of a approximately 40-kb cosmid insert can be obtained by a single packaging reaction, and its restriction map can be simultaneously obtained.

Bacteriophage T3↗
PubMed2005

In vitro DNA packaging of PRD1: a common mechanism for internal-membrane viruses.

PRD1 is the type virus of the Tectiviridae family. Its linear double-stranded DNA genome has covalently attached terminal proteins and is surrounded by a membrane, which is further enclosed within an icosahedral protein capsid. Similar to tailed bacteriophages, PRD1 packages its DNA into a preformed procapsid. The PRD1 putative packaging ATPase P9 is a structural protein located at a unique vertex of the capsid. An in vitro system for packaging DNA into preformed empty procapsids was developed. The system uses cell extracts of overexpressed P9 protein and empty procapsids from a P9-deficient mutant virus infection and PRD1 DNA containing a LacZalpha-insert. The in vitro packaged virions produce distinctly blue plaques when plated on a suitable host. This is the first time that a viral genome is packaged in vitro into a membrane vesicle. Comparison of PRD1 P9 with putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses revealed a new packaging ATPase-specific motif. Surprisingly the viruses having this packaging ATPase motif, and thus considered to be related, were the same as those recently grouped together using the coat protein fold and virion architecture. Our finding here strongly supports the idea that all these viruses infecting hosts in all domains of life had a common ancestor.

Adenosine Triphosphatases↗
PubMed2006

Controlling bacteriophage phi29 DNA-packaging motor by addition or discharge of a peptide at N-terminus of connector protein that interacts with pRNA.

Bacteriophage phi29 utilizes a motor to translocate genomic DNA into a preformed procapsid. The motor contains six pRNAs, an enzyme and one 12-subunit connector with a central channel for DNA transportation. A 20-residue peptide containing a His-tag was fused to the N-terminus of the connector protein gp10. This fusion neither interfered with procapsid assembly nor affected the morphology of the prolate-shaped procapsid. However, the pRNA binding and virion assembly activity were greatly reduced. Such decreased functions can be switched back on by the removal of the tag via protease cleavage, supporting the previous finding that the N-terminus of gp10 is essential for the pRNA binding. The DNA-packaging efficiency with dimeric pRNA was more seriously affected by the extension than with monomeric pRNA. It is speculated that the fusion of the tag generated physical hindrance to pRNA binding, with greater influence for the dimers than the monomers due to their size. These results reveal a potential to turn off and turn on the motor by attaching or removing, respectively, a component to outer part of the motor, and offers an approach for the inhibition of viral replication by using a drug or a small peptide targeted to motor components.

Bacillus Phages↗
PubMed1991

Analysis of interactions among factors involved in the bacteriophage T3 DNA packaging reaction in a defined in vitro system.

During head assembly of phage T3, DNA is packaged into the cavity of a preformed protein shell, called the prohead, with the aid of noncapsid, packaging proteins, the products of genes 18 and 19 (gp18 and gp19). gp18 and gp19 separately form complexes with DNA and proheads, respectively. These complexes associate to form a precursor which can be converted to filled heads by the addition of ATP. Interactions among factors involved in DNA packaging were analyzed. In the presence of ATP, gp19 formed functional complexes with proheads. Formation of gp19-prohead complex showed a sigmoidal dependence on ATP concentration with a half maximal concentration of about 7.5 microM. Six molecules of gp19 bound to the prohead at a saturating amount of gp19. gp19 did not bind to proheads lacking the connector of gp8 (8- prohead). In the absence of ATP, proheads were inactivated by gp19. The gp19-prohead complexes formed in the absence of ATP contained 20-30 gp19 molecules per prohead and formed multimeric aggregates. 8- proheads did not bind gp19 and did not form such aggregates even in the absence of ATP. From these results, we conclude that 6 molecules of gp19 bind to the gp8 connector structure in the portal vertex of the prohead. The cleavage patterns of gp19 by several proteases were altered by the addition of ATP, indicating that ATP induces a conformational change in gp19, gp18 bound only to linear, duplex DNA.

Adenosine Triphosphate↗
PubMed1984

Initiation by bacteriophage T1 of DNA packaging at a site between the P and Q genes of bacteriophage lambda.

The growth of phage T1 on cells tandemly lysogenic for heteroimmune lambdoid prophages leads to a nonrandom packaging of lambda DNA by T1. A site, called esp-lambda, is located between the P and Q genes of lambda and results in increased packaging to the left by T1. When cloned into pBR322, the esp-lambda site causes a significant increase in transduction of the plasmid by T1. The nin5 deletion inactivates esp-lambda.

Bacteriophage lambda↗
PubMed2003

Bacillus subtilis bacteriophage SPP1 DNA packaging motor requires terminase and portal proteins.

Initiation of headful packaging of SPP1 DNA concatemers involves the interaction of the terminase, G1P and G2P, and the portal protein, G6P. G1P, which specifically recognizes the non-adjacent pacL and pacR subsites and directs loading of G2P to pacC, interacts with G6P. G2P, which has endonuclease, DNA binding, and ATPase activities, interacts with G1P and does it transiently with G6P. The stoichiometry of G1P on the G1P.G2P complex promotes the transition from a G2P endonuclease to an ATPase. G6P does not alter the endonuclease activity of G2P. Both G1P and G6P, which do not have endogenous ATPase activity, synergistically enhance and modulate the ATPase activity of G2P. Based on these results, we propose a model in which the modulation of the ATPase and endonuclease activities of G2P accounts for the role of the terminase in headful packaging.

Bacillus subtilis↗
PubMed2002

DNA packaging: a new class of molecular motors.

DNA is packaged into preformed bacteriophage capsids to liquid crystalline density by the action of a portal protein complex. Single molecule packaging studies indicate that this is a new and extremely powerful class of molecular motors.

Capsid↗
PubMed2003

Construction of phi29 DNA-packaging RNA monomers, dimers, and trimers with variable sizes and shapes as potential parts for nanodevices.

Recently, DNA and RNA have been under extensive scrutiny with regard to their feasibility as parts in nanotechnology. The DNA-packaging motor of bacterial virus phi29 contains six copies of pRNA molecules, which together form a hexameric ring as a crucial part of the motor. This ring is formed via hand-in-hand interaction by Watson-Crick base pairing of four nucleotides from the left and right loops. Here we report that this pRNA tends to form a circular ring by hand-in-hand contact even when in dimer or trimer form, thus implying that the pRNA structure is flexible. Stable dimers and trimers have been formed from the monomer unit in a protein-free environment with nearly 100% efficiency. The dimers and trimers could be isolated by density gradient sedimentation or purified from native gel. Dimers and trimers were resistant to pH levels as low as 4 and as high as 10, to temperatures as low as -70 degrees C and as high as 80 degrees C, and to high salt concentrations such as 2 M NaCl and 2 M MgCl2. Further study showed that pRNA dimers or trimers with variable lengths could be constructed. Seventy-five bases were found to be the central component in this formation. The elongation of RNA at the 3' end up to 120 bases did not hinder their formation. Other conditions, including the salt requirement for the formation of monomers, dimers, and trimers, have been investigated. RNA monomers, dimers, and trimers with variable lengths are potential parts for nanodevices.

Bacillus Phages↗
PubMed2000

Differences in DNA packaging genes and sensitivity to benzimidazole ribonucleosides between human cytomegalovirus strains AD169 and Towne.

The AD169 strain of human cytomegalovirus was approximately twofold more sensitive to polyhalogenated benzimidazole ribonucleosides than Towne strain. Sequence differences between the two strains have been identified in genes UL51, UL52, UL56, UL77, UL89 and UL104. Because these genes are involved in cleavage and packaging of viral DNA and the benzimidazole ribonucleosides inhibit this process, these sequence differences may be involved in the difference in drug sensitivity.

Benzimidazoles↗
PubMed1996

An improved cosmid vector for the nested deletion method using the bacteriophage T3 DNA packaging system.

We constructed a new cosmid vector suitable for the previously developed nested deletion method which used the in vitro DNA packaging system of bacteriophage T3. The first step of this method is linearization of a cosmid clone to be packaged, and we previously introduced cleavage at the cos site using lambda-Terminase, but optimization of the reaction conditions was required for complete digestion because of its instability. In the newly constructed vector, pAT5, the sites of 4 different restriction enzymes, Sse8387I, Asc I, Fse I and Pme I, each of which recognizes an 8-bp sequence (8-base cutter) were introduced in the vicinity of the cos site. In addition, the species of restriction sites for cloning were increased to broaden its application. The cosmid clone constructed by this new vector could be linearized at one of the 8-base cutter sites which are assumed to rarely occur in the genome, and followed by in vitro packaging, nested deletion clones were successfully prepared.

Bacteriophage T3↗
PubMed2005

Identification of an interacting coat-external scaffolding protein domain required for both the initiation of phiX174 procapsid morphogenesis and the completion of DNA packaging.

The phiX174 external scaffolding protein D mediates the assembly of coat protein pentamers into procapsids. There are four external scaffolding subunits per coat protein. Organized as pairs of asymmetric dimers, the arrangement is unrelated to quasi-equivalence. The external scaffolding protein contains seven alpha-helices. The protein's core, alpha-helices 2 to 6, mediates the vast majority of intra- and interdimer contacts and is strongly conserved in all Microviridae (canonical members are phiX174, G4, and alpha3) external scaffolding proteins. On the other hand, the primary sequences of the first alpha-helices have diverged. The results of previous studies with alpha3/phiX174 chimeric external scaffolding proteins suggest that alpha-helix 1 may act as a substrate specificity domain, mediating the initial coat scaffolding protein recognition in a species-specific manner. However, the low sequence conservation between the two phages impeded genetic analyses. In an effort to elucidate a more mechanistic model, chimeric external scaffolding proteins were constructed between the more closely related phages G4 and phiX174. The results of biochemical analyses indicate that the chimeric external scaffolding protein inhibits two morphogenetic steps: the initiation of procapsid formation and DNA packaging. phiX174 mutants that can efficiently utilize the chimeric protein were isolated and characterized. The substitutions appear to suppress both morphogenetic defects and are located in threefold-related coat protein sequences that most likely form the pores in the viral procapsid. These results identify coat-external scaffolding domains needed to initiate procapsid formation and provide more evidence, albeit indirect, that the pores are the site of DNA entry during the packaging reaction.

Amino Acid Sequence↗
PubMed2005

A-tract clusters may facilitate DNA packaging in bacterial nucleoid.

Molecular mechanisms of bacterial chromosome packaging are still unclear, as bacteria lack nucleosomes or other apparent basic elements of DNA compaction. Among the factors facilitating DNA condensation may be a propensity of the DNA molecule for folding due to its intrinsic curvature. As suggested previously, the sequence correlations in genome reflect such a propensity [Trifonov and Sussman (1980) Proc. Natl Acad. Sci. USA, 77, 3816-3820]. To further elaborate this concept, we analyzed positioning of A-tracts (the sequence motifs introducing the most pronounced DNA curvature) in the Escherichia coli genome. First, we observed that the A-tracts are over-represented and distributed 'quasi-regularly' throughout the genome, including both the coding and intergenic sequences. Second, there is a 10-12 bp periodicity in the A-tract positioning indicating that the A-tracts are phased with respect to the DNA helical repeat. Third, the phased A-tracts are organized in approximately 100 bp long clusters. The latter feature was revealed with the help of a novel approach based on the Fourier series expansion of the A-tract distance autocorrelation function. Since the A-tracts introduce local bends of the DNA duplex and these bends accumulate when properly phased, the observed clusters would facilitate DNA looping. Also, such clusters may serve as binding sites for the nucleoid-associated proteins that have affinities for curved DNA (such as HU, H-NS, Hfq and CbpA). Therefore, we suggest that the approximately 100 bp long clusters of the phased A-tracts constitute the 'structural code' for DNA compaction by providing the long-range intrinsic curvature and increasing stability of the DNA complexes with architectural proteins.

AT Rich Sequence↗
PubMed1982

DNA packaging in vitro by an isolated bacteriophage T7 procapsid.

The results of previous in vivo studies indicate that a DNA-free procapsid (capsid I) packages bacteriophage T7 DNA during infection of Escherichia coli. It was shown here that capsid I, isolated by electrophoresis in metrizamide density gradients, packaged DNA and formed infectious phage particles when incubated in vitro with extracts deficient in capsid proteins.

Capsid↗