Role of RNA in bacteriophage phi 29 DNA packaging

A novel bacteriophage phi 29 RNA of 174 nucleotides is essential for the in vitro packaging of the DNA-terminal protein complex into proheads. The RNA, bound to the prohead portal vertex (connector), participates in assembly and function of the DNA translocating ATPase and in recognition of the DNA left-end during the course of the packaging reaction. The RNA is present in related phages and varies widely in primary sequence, but its secondary structure, as deduced by phylogenetic analysis, is both highly conserved and unique among small RNAs.     

Cleaving the prohead RNA of bacteriophage phi 29 alters the in vitro packaging of restriction fragments of DNA-gp3

In vitro packaging of restriction fragments of the bacteriophage phi 29 DNA-gp3 (DNA-gene product 3 complex) in the defined system was dependent on prohead RNA. Truncated prohead RNAs were obtained by in situ RNase A digestion, isolated and sequenced. Proheads having the intact 174 base RNA were compared to proheads having RNAs of 120, 95, 71, 69 or 54 bases for the capacity to package the DNA-gp3 left and right ends and internal (non-end) fragments generated by the restriction enzymes EcoRI, HpaI and BstNI. Proheads with the 174 or 120 base RNAs packaged both left and right ends; internal fragments were packaged more efficiently by proheads with the 120 base RNA. Proheads with the 95 base RNA packaged DNA-gp3 left ends and internal fragments efficiently, but lost the capacity to package right ends. Only internal fragments were packaged by proheads with the 71 base RNA, and proheads having 69 or 54 base RNAs were inactive. RNA-free proheads were effectively reconstituted with purified 174 and 120 base RNAs to produce particles similar in biological activity to the proheads from which the RNAs were isolated. The 95 base RNA was the smallest RNA of the group that could reconstitute the prohead and direct fragment packaging, although packaging was inefficient. Alteration of the specificity of DNA fragment packaging with truncated prohead RNAs has delineated RNA domains that function in DNA-gp3 recognition and prohead binding.     

RNA dependence of the bacteriophage phi 29 DNA packaging ATPase.

The activity of the DNA packaging adenosine triphosphatase (ATPase) of the Bacillus subtilis bacteriophage phi 29 is dependent upon prohead RNA. The 174 nucleotide viral-encoded RNA is positioned on the head-tail connector at the portal vertex of the phi 29 precursor shell (prohead). Here, the RNA interacts with the ATP-binding gene 16 product (gp16) to constitute the DNA-packaging ATPase and initiate DNA packaging in vitro. Both the prohead connector (gene 10 product, gp10) and gp16 may utilize an RNA recognition motif characteristic of a number of RNA-associated proteins, and the binding of gp16 by proheads shields the prohead RNA from RNase A. The ATPase activity of gp16 is stimulated fourfold by RNA and tenfold by proheads with RNA. RNA is needed continuously for the gp16/RNA ATPase activity and is essential for the gp16/prohead ATPase activity. The prohead, with its connector, RNA and associated gp16 in an assembly-regulated configuration, hydrolyzes ATP and drives phi 29 DNA translocation.     

Characterization of the small RNA of the bacteriophage phi 29 DNA packaging machine.

The prohead connector of the bacteriophage luminal diameter 29 DNA packaging machine was reconstructed with the small RNA that regulates DNA packaging in vitro. The complete sequence of the 120 nucleotide RNA proved its origination from the promoter PE1(A1) of the left early region of phi 29 DNA, the end packaged first during assembly. The prohead RNA was clearly distinct from eubacterial 5S rRNA in sequence and composition.     

Conservation of the capsid structure in tailed dsDNA bacteriophages: the pseudoatomic structure of phi29

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.     

Mutant prohead RNAs in the in vitro packaging of bacteriophage phi 29 DNA-gp3.

The 174-base prohead RNA encoded by bacteriophage phi 29 of Bacillus subtilis, essential for packaging of the DNA-gp3 (DNA-gene product 3) complex, was expressed efficiently from the cloned gene. Computer programs for RNA structure analysis were used to fold hypothetical RNA mutants and thus to target mutagenesis of the RNA for studies of structure and function. Five mutants of the RNA were then produced by oligonucleotide-directed mutagenesis that were altered in the primary sequence at selected sites; two of these mutants were predicted to be altered in secondary structure from a model established previously by a phylogenetic analysis. The binding of the 32P end-labeled mutant RNAs to RNA-free proheads was comparable with that of the wild-type RNA. However, the capability of the mutant RNAs to reconstitute RNA-free proheads for DNA-gp3 packaging in the defined in vitro system and for assembly of phage in RNA-free extracts was variable, depending upon the alteration. Changes of highly conserved bases that retained the predicted secondary structure of the RNA model were tolerated to a much greater extent than changes predicted to alter the RNA secondary structure.     

DNA packaging motor assembly intermediate of bacteriophage phi29

Unraveling the structure and assembly of the DNA packaging ATPases of the tailed double-stranded DNA bacteriophages is integral to understanding the mechanism of DNA translocation. Here, the bacteriophage phi29 packaging ATPase gene product 16 (gp16) was overexpressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the phi29 precursor capsid (prohead) to produce a packaging motor intermediate that was fully active in in vitro DNA packaging. The formation of higher oligomers of the gp16 from monomers was concentration dependent and was characterized by analytical ultracentrifugation, gel filtration, and electron microscopy. The binding of multiple copies of gp16 to the prohead was dependent on the presence of an oligomer of 174- or 120-base prohead RNA (pRNA) fixed to the head-tail connector at the unique portal vertex of the prohead. The use of mutant pRNAs demonstrated that gp16 bound specifically to the A-helix of pRNA, and ribonuclease footprinting of gp16 on pRNA showed that gp16 protected the CC residues of the CCA bulge (residues 18-20) of the A-helix. The binding of gp16 to the prohead/pRNA to constitute the complete and active packaging motor was confirmed by cryo-electron microscopy three-dimensional reconstruction of the prohead/pRNA/gp16 complex. The complex was capable of supercoiling DNA-gp3 as observed previously for gp16 alone; therefore, the binding of gp16 to the prohead, rather than first to DNA-gp3, represents an alternative packaging motor assembly pathway.     

Diverse self-association properties within a family of phage packaging RNAs

The packaging RNA (pRNA) found in phi29 bacteriophage is an essential component of a molecular motor that packages the phage''s DNA genome. The pRNA forms higher-order multimers by intermolecular “kissing” interactions between identical molecules. The phi29 pRNA is a proven building block for nanotechnology and a model to explore the rare phenomenon of naturally occurring RNA self-association. Although the self-association properties of the phi29 pRNA have been extensively studied and this pRNA is used in nanotechnology, the characteristics of phylogenetically related pRNAs with divergent sequences are comparatively underexplored. These diverse pRNAs may lend new insight into both the rules governing RNA self-association and for RNA engineering. Therefore, we used a combination of biochemical and biophysical methods to resolve ambiguities in the proposed secondary structures of pRNAs from M2, GA1, SF5, and B103 phage, and to discover that different naturally occurring pRNAs form multimers of different stoichiometry and thermostability. Indeed, the M2 pRNA formed multimers that were particularly thermostable and may be more useful than phi29 pRNA for many applications. To determine if diverse pRNA behaviors are conferred by different kissing loop sequences, we designed and tested chimeric RNAs based on our revised secondary structural models. We found that although the kissing loops are essential for self-association, the critical determinant of multimer stability and stoichiometry is likely the diverse three-way junctions found in these RNAs. Using known features of RNA three-way junctions and solved structures of phi29 pRNA''s junction, we propose a model for how different junctions affect self-association.     

Prohead and DNA-gp3-dependent ATPase activity of the DNA packaging protein gp16 of bacteriophage phi 29

The ATPase activity of the DNA packaging protein gp16 (gene product 16) of bacteriophage phi 29 was studied in the completely defined in-vitro assembly system. ATP was hydrolyzed to ADP and Pi in the packaging reaction that included purified proheads, DNA-gp3 and gp16. Approximately one molecule of ATP was used in the packaging of 2 base-pairs of phi 29 DNA, or 9 X 10(3) ATP molecules per virion. The hydrolysis of ATP by gp16 was both prohead and DNA-gp3 dependent. gp16 contained both the "A-type" and the "B-type" ATP-binding consensus sequences (Walker et al., 1982) and the predicted secondary structure for ATP binding. The A-type sequence of gp16 was "basic-hydrophobic region-G-X2-G-X-G-K-S-X7-hydrophobic", and similar sequences were found in the phage DNA packaging proteins gpA of lambda, gp19 of T7 and gp17 of T4. Having both the ATP-binding and potential magnesium-binding domains, all of these proteins probably function as ATPases and may have common prohead-binding capabilities. The phi 29 protein gp3, covalently bound to the DNA, may be analogous in function to proteins gpNul of lambda and gpl of phi 21 that bind the DNA.     

RNA-mediated specificity of DNA packaging into hybrid lambda/phi 29 proheads.

A small RNA (pRNA, 174 nt) is known to be essential for DNA packaging in bacteriophage phi 29. However, in an in vitro DNA packaging system based on hybrid lambda/phi 29 proheads (made up of head proteins from phage lambda and connectors from phage phi 29), the specificity of DNA packaging is lost, and different RNA molecules fulfil the requirements for DNA packaging, albeit with less efficiency than phi 29 pRNA. Competition assays with RNAs from different sources have shown that phi 29 connectors bind preferentially pRNA. An increase in the efficiency of phi 29 DNA packaging into hybrid proheads induced by phi 29 pRNA is observed because, when phi 29 pRNA is incubated with hybrid proheads, phi 29 DNA is packaged more efficiently than other DNAs of similar length. Furthermore, when hybrid proheads carrying phi 29 pRNA are incubated with a mixture of DNAs from different sources, phi 29 DNA is selectively packaged, thus indicating that phi 29 pRNA determines the specificity of DNA packaging.     

Initiation events in in-vitro packaging of bacteriophage phi 29 DNA-gp3

Initiation events in the packaging of bacteriophage phi 29 DNA-gp3 (DNA-gene product 3 complex) were studied in a completely defined in-vitro system that included purified proheads, DNA-gp3 and the DNA packaging protein gp16. In the sequential interactions, gp16 first bound to, and was modified by, the prohead. The prohead-gp16 complex then bound to DNA-gp3, resulting in a second modification of gp16 that permitted binding of ATP. DNA-gp3 aggregates were produced, and the hydrolysis of ATP accompanied DNA-gp3 packaging. Binding and hydrolysis of ATP by gp16 was both prohead- and DNA-gp3-dependent. Interruption of packaging by DNase I addition revealed filled heads but few particles containing partial lengths of DNA, suggesting that following a rate-limiting initiation, the translocation of DNA-gp3 into the prohead was much faster in the defined in-vitro system than in extracts.     

Role of the amino-terminal domain of bacteriophage phi 29 connector in DNA binding and packaging.

The connector of bacteriophage phi 29 is required for prohead assembly, binds DNA, and drives DNA packaging into viral proheads. Limited proteolysis of the connector protein with endoproteinase Glu-C from Staphylococcus aureus V8 and chymotrypsin showed that a domain of the NH2-terminal region is involved in DNA binding and in the subsequent packaging into preformed proheads, but not in prohead assembly. Mutants in specific amino acids of the NH2-terminal domain, obtained by directed mutagenesis techniques, showed that the Ala1-Arg2-Lys3-Arg4 region of the connector is absolutely necessary for DNA packaging into the proheads as well as for efficient DNA binding.     

Topology of the components of the DNA packaging machinery in the phage phi29 prohead

Chromosome condensation inside dsDNA viral particles is a complex process requiring the coordinated action of several viral components. The similarity of the process in different viral systems has led to the suggestion that there is a common underlying mechanism for DNA packaging, in which the portal vertex or connector plays a key role. We have studied the topology of the packaging machinery using a number of antibodies directed against different domains of the connector. The charged amino-terminal, the carboxyl-terminal, and the RNA binding domain are accessible areas in the connector assembled into the prohead, while the domains corresponding to the 12 large appendages of the connector are buried inside the prohead. Furthermore, while the antibodies against the carboxyl and amino-terminal do not affect the packaging reaction, incubation of proheads with antibodies against the RNA binding domain abolishes the packaging activity. The comparison of the three-dimensional reconstructions of bacteriophage phi29 proheads with proheads devoid of their specific pRNA by RNase treatment shows that this treatment removes structural elements of the distal vertex of the portal structure, suggesting that the pRNA required for packaging is located at the open gate of the channel in the narrow side of the connector.     

Implication of the prohead RNA in phage phi29 DNA packaging

RNA is an important component of many biological processes, including DNA encapsidation of bacteriophage phi29 of Bacillus subtilis. Interestingly, the prohead RNA is involved in this encapsidation, and was found in monomer, dimer, pentamer and hexamer conformations. This article presents and debates current knowledge about the prohead RNA structures, mechanisms, and roles in DNA encapsidation. A new dimer structure is presented, and its specific role in DNA encapsidation is discussed.     

Confirmation of the helical structure of the 5'/3' termini of the essential DNA packaging pRNA of phage phi 29.

Bacteriophage phi 29 is typical of double-stranded DNA viruses in that its genome is packaged into a preformed procapsid during viral assembly. An intriguing feature of phi 29 is the presence of a 120-base virus-encoded RNA (pRNA) that is indispensable for DNA packaging. Phylogenetic comparison of similar RNAs in numerous phages has revealed that the secondary structure of the pRNA is well conserved. Computer analysis predicts the presence of an extensive segment of helix with three single-base bulges generated by the pairing of the 5' and 3' ends. The desire to understand the role played by the pRNA in DNA packaging has led to a mutational analysis of the 5'-/3'-terminal region, which is believed to be important in DNA translocation. Deletion of 3 bases from the 3' end of the RNA, shortening the pRNA from 120 to 117 bases, was tolerated without loss of activity, but additional deletion of the base 117 resulted in 100-fold less activity, and a 115-base pRNA was virtually nonfunctional. Additionally, the three unpaired one-base bulges within the helical stretches of the paired proximate ends were nonessential for pRNA activity, as demonstrated by deletion of the bulge individually. An extensive series of helix disruptions by single- and multiple-base substitution almost invariably led to the loss of DNA packaging activity. Additional mutations that restored predicted base pairings rescued pRNA activity. This second site suppression confirmed that the 5'- and 3'-end region was paired and was indeed a helical stretch. The secondary structure was of greater importance than the primary sequence, with the exception of the requirement of an adenine at either the third or fourth position. The specific requirement of an adenine in phi 29 pRNA at this position, as well as conservation of this position in other phage pRNAs, implicates that this base may play a special role in either the DNA-packaging reaction or the maintenance of the pRNA tertiary structure.     

Defining molecular and domain boundaries in the bacteriophage phi29 DNA packaging motor

Cryo-electron microscopy (cryo-EM) studies of the bacteriophage phi29 DNA packaging motor have delineated the relative positions and molecular boundaries of the 12-fold symmetric head-tail connector, the 5-fold symmetric prohead RNA (pRNA), the ATPase that provides the energy for packaging, and the procapsid. Reconstructions, assuming 5-fold symmetry, were determined for proheads with 174-base, 120-base, and 71-base pRNA; proheads lacking pRNA; proheads with ATPase bound; and proheads in which the packaging motor was missing the connector. These structures are consistent with pRNA and ATPase forming a pentameric motor component around the unique vertex of proheads. They suggest an assembly pathway for the packaging motor and a mechanism for DNA translocation into empty proheads.     

Cryoelectron-microscopy image reconstruction of symmetry mismatches in bacteriophage phi29

A method has been developed for three-dimensional image reconstruction of symmetry-mismatched components in tailed phages. Although the method described here addresses the specific case where differing symmetry axes are coincident, the method is more generally applicable, for instance, to the reconstruction of images of viral particles that deviate from icosahedral symmetry. Particles are initially oriented according to their dominant symmetry, thus reducing the search space for determining the orientation of the less dominant, symmetry-mismatched component. This procedure produced an improved reconstruction of the sixfold-symmetric tail assembly that is attached to the fivefold-symmetric prolate head of phi29, demonstrating that this method is capable of detecting and reconstructing an object that included a symmetry mismatch. A reconstruction of phi29 prohead particles using the methods described here establishes that the pRNA molecule has fivefold symmetry when attached to the prohead, consistent with its proposed role as a component of the stator in the phi29 DNA packaging motor.     

Alanine scanning and Fe-BABE probing of the bacteriophage ø29 prohead RNA-connector interaction

The DNA packaging motor of the Bacillus subtilis bacteriophage ?29 prohead is comprised in part of an oligomeric ring of 174 base RNA molecules (pRNA) positioned near the N termini of subunits of the dodecameric head-tail connector. Deletion and alanine substitution mutants in the connector protein (gp10) N terminus were assembled into proheads in Escherichia coli and the particles tested for pRNA binding and DNA-gp3 packaging in vitro. The basic amino acid residues RKR at positions 3-5 of the gp10 N terminus were central to pRNA binding during assembly of an active DNA packaging motor. Conjugation of iron(S)-1-(p-bromoacetamidobenzyl) ethylenediaminetetraacetate (Fe-BABE) to residue S170C in the narrow end of the connector, near the N terminus, permitted hydroxyl radical probing of bound [(32)P]pRNA and identified two discrete sites proximal to this residue: the C-helix at the junction of the A, C and D helices, and the E helix and the CE loop/D loop of the intermolecular base pairing site.     

The three-dimensional structure of a DNA translocating machine at 10 A resolution.

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.