Mutational analysis of the primer RNA template region in the replication origin (oric) of bacteriophage G4: priming signal recognition by Escherichia coli primase

The primase-dependent phage G4 origin of complementary DNA strand synthesis (G4oric) contains three stable stem-loops (I, II, and III) upstream from the initiation point of primer RNA (pRNA). Site-directed mutagenesis was used to introduce alterations into the nucleotide (nt) sequence of the G4oric pRNA template region. Mutations in stem-loop I, that changed the length of the stem and the sequence of the loop, slightly depressed, but did not abolish, G4oric activity. However, functional G4oric activity was destroyed when the sequence containing the starting position of pRNA synthesis was deleted, or when insertions were introduced between the pRNA starting position (5'-CTG-3') and stem-loop I. Reintroducing a CTG as part of a PstI linker close to stem-loop I, however, resulted in recovery of G4oric functional activity. These results suggest that the specific nt sequence, containing 5'-CTG-3', between nt 3994 and 4007, and also the distance between the starting position of pRNA synthesis and stem-loop I, are essential structural features for G4oric function.     

Role of the CCA bulge of prohead RNA of bacteriophage ø29 in DNA packaging

The oligomeric ring of prohead RNA (pRNA) is an essential component of the ATP-driven DNA packaging motor of bacteriophage ?29. The A-helix of pRNA binds the DNA translocating ATPase gp16 (gene product 16) and the CCA bulge in this helix is essential for DNA packaging in vitro. Mutation of the bulge by base substitution or deletion showed that the size of the bulge, rather than its sequence, is primary in DNA packaging activity. Proheads reconstituted with CCA bulge mutant pRNAs bound the packaging ATPase gp16 and the packaging substrate DNA-gp3, although DNA translocation was not detected with several mutants. Prohead/bulge-mutant pRNA complexes with low packaging activity had a higher rate of ATP hydrolysis per base pair of DNA packaged than proheads with wild-type pRNA. Cryoelectron microscopy three-dimensional reconstruction of proheads reconstituted with a CCA deletion pRNA showed that the protruding pRNA spokes of the motor occupy a different position relative to the head when compared to particles with wild-type pRNA. Therefore, the CCA bulge seems to dictate the orientation of the pRNA spokes. The conformational changes observed for this mutant pRNA may affect gp16 conformation and/or subsequent ATPase-DNA interaction and, consequently, explain the decreased packaging activity observed for CCA mutants.     

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.     

The procapsid binding domain of phi29 packaging RNA has a modular architecture and requires 2'-hydroxyl groups in packaging RNA interaction

The phi29 packaging RNA (pRNA) is an essential component in the phi29 bacteriophage DNA packaging motor, the strongest biomolecular motor known today. Utilizing Mg2+-dependent intermolecular base pairing interactions between two 4-nucleotide loops within the pRNA procapsid binding domain, multiple copies of pRNA form a ring-shaped complex that is indispensable for packaging motor function. To understand pRNA structural organization and pRNA/pRNA interaction, studies were carried out on pRNA closed dimers, the simplest functional pRNA complex believed to be the building blocks for assembling the oligomeric ring. Tertiary folding and interactions in various pRNA mutants were evaluated based on measured closed dimer affinity that is directly linked to the proper positioning of the interacting loops. The data revealed that the procapsid binding domain contains two autonomous modules that are capable of interacting noncovalently to form a fully active species in pRNA/pRNA interaction. Deleting the 2'-hydroxyl groups in one of the interacting loops weakens the dimer affinity by 125-fold, suggesting potential tertiary interactions involving these 2'-hydroxyl groups. The results provide evidence that nonbase functional groups are involved in pRNA folding and interaction and lead to a simple model that describes the pRNA monomer configuration in terms of three arms spanning a hinge. The functional constructs developed here will aid biophysical and biochemical investigations of pRNA structure and function, as well as developments of pRNA-based technology for nanoscience and gene therapy.     

Use of circular permutation to assess six bulges and four loops of DNA-packaging pRNA of bacteriophage phi29.

A 120-base phage phi29 encoded RNA (pRNA) has a novel role in DNA packaging. This pRNA possesses five single-base bulges, one three-base bulge, one bifurcation bulge, one bulge loop, and two stem loops. Circularly permuted pRNAs (cpRNA) were constructed to examine the function of these bulges and loops as well as their adjacent sequences. Each of the five single-base bulges was nonessential. The bifurcation bulge could be deleted and replaced with a new opening to provide flexibility for maintaining an overall correct folding in three-way junction. All of these nonessential bulges or their adjacent bases could be used as new termini for cpRNAs. The three-base (C18C19A20) bulge was dispensable for procapsid binding, but was indispensable for DNA packaging. The secondary structure around this CCA bulge and the phylogenetically conserved bases within or around it were investigated. Bases A14C15U16 were confirmed, by compensatory modification, to pair with U103G102A101. A99 was needed only to allow the proper folding of CCA bulge in the appropriate sequence order and distance constraints. Beyond these, the seemingly phylogenetic conservation of other bases has little role in pRNA activity. Each of the three stem loops was essential for procapsid binding, DNA packaging, and phage assembly. Disruption of the middle of any one of the loops resulted in dramatic reductions in procapsid binding, subsequent DNA packaging, and phage assembly activities. However, disruption of the loops at sequences that were close to double-stranded regions of the RNA did not interfere with pRNA activity significantly. Our results suggest that double-stranded helical regions near these loops were most likely not involved in interactions with components of the DNA-packaging machinery. Instead, these regions appear to be merely present to serve as a scaffolding to display the single-stranded loops that are important for pRNA tertiary structure or for interaction with the procapsid or other packaging components.     

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.     

The Contribution of Peripheral Stem-Loops to the Catalytic Activity of Archaeal RNase P RNA from Pyrococcus horikoshii OT3

We investigated the contribution of peripheral stem-loops to the catalytic activity of an archaeal RNase P RNA, PhopRNA, from Pyrococcus horikoshii OT3. PhopRNA mutants, in which the stem-loops were individually deleted, were prepared and characterized with respect to precursor tRNA (pre-tRNA) cleavage activity in the presence of five RNase P proteins. All the mutants retained the activity to some extent, indicating that they are moderately implicated in catalysis. Further characterization suggested that the stem-loops serve largely as binding sites for the proteins, and that their interactions are predominantly involved in stabilization of the active conformation of PhopRNA.     

Complete inhibition of virion assembly in vivo with mutant procapsid RNA essential for phage phi 29 DNA packaging.

A highly efficient method for the inhibition of bacteriophage phi 29 assembly was developed with the use of mutant forms of the viral procapsid (or packaging) RNA (pRNA) indispensable for phi 29 DNA packaging. Phage phi 29 assembly was severely reduced in vitro in the presence of mutant pRNA and completely blocked in vivo when the host cell expressed mutant pRNA. Addition of 45% mutant pRNA resulted in a reduction of infectious virion production by 4 orders of magnitude, indicating that factors involved in viral assembly can be targets for efficient and specific antiviral treatment. The mechanism leading to the high efficiency of inhibition was attributed to two pivotal features. First, the pRNA contains two separate, essential functional domains, one for procapsid binding and the other for a DNA-packaging role other than procapsid binding. Mutation of the DNA-packaging domain resulted in a pRNA with no DNA-packaging activity but intact procapsid binding competence. Second, multiple copies of the pRNA were involved in the packaging of one genome. This higher-order dependence of pRNA in viral replication concomitantly resulted in its higher-order inhibitory effect. This finding suggested that the collective DNA-packaging activity of multiple copies of pRNA could be disrupted by the incorporation of perhaps an individual mutant pRNA into the group. Although this mutant pRNA could not be used for the inhibition of the replication of other viruses directly, the principle of using molecules with two functional domains and multiple-copy involvement as targets for antiviral agents could be applied to certain viral structural proteins, enzymes, and other factors or RNAs involved in the viral life cycle. This principle also implies a strategy for gene therapy, intracellular immunization, or construction of transgenic plants resistant to viral infection.     

Affinity of molecular interactions in the bacteriophage phi29 DNA packaging motor

DNA packaging in the bacteriophage 29 involves a molecular motor with protein and RNA components, including interactions between the viral connector protein and molecules of pRNA, both of which form multimeric complexes. Data are presented to demonstrate the higher order assembly of pRNA together with the affinity of pRNA:pRNA and pRNA:connector interactions, which are used to propose a model for motor function. In solution, pRNA can form dimeric and trimeric multimers in a magnesium-dependent manner, with dissociation constants for multimerization in the micromolar range. pRNA:connector binding is also facilitated by the presence of magnesium ions, with a nanomolar apparent dissociation constant for the interaction. From studies with a mutant pRNA, it appears that multimerization of pRNA is not essential for connector binding and it is likely that connector protein is involved in the stabilization of higher order RNA multimers. It is proposed that magnesium ions may promote conformational change that facilitate pRNA:connector interactions, essential for motor function.     

Interaction of gp16 with pRNA and DNA for genome packaging by the motor of bacterial virus phi29

One striking feature in the assembly of linear double-stranded (ds) DNA viruses is that their genome is translocated into a preformed protein coat via a motor involving two non-structural components with certain characteristics of ATPase. In bacterial virus phi29, these two components include the protein gp16 and a packaging RNA (pRNA). The structure and function of other phi29 motor components have been well elucidated; however, studies on the role of gp16 have been seriously hampered by its hydrophobicity and self-aggregation. Such problems caused by insolubility also occur in the study of other viral DNA-packaging motors. Contradictory data have been published regarding the role and stoichiometry of gp16, which has been reported to bind every motor component, including pRNA, DNA, gp3, DNA-gp3, connector, pRNA-free procapsid, and procapsid/pRNA complex. Such conflicting data from a binding assay could be due to the self-aggregation of gp16. Our recent advance to produce soluble and highly active gp16 has enabled further studies on gp16. It was demonstrated in this report that gp16 bound to DNA non-specifically. gp16 bound to the pRNA-containing procapsid much more strongly than to the pRNA-free procapsid. The domain of pRNA for gp16 interaction was the 5'/3' paired helical region. The C18C19A20 bulge that is essential for DNA packaging was found to be dispensable for gp16 binding. This result confirms the published model that pRNA binds to the procapsid with its central domain and extends its 5'/3' DNA-packaging domain for gp16 binding. It suggests that gp16 serves as a linkage between pRNA and DNA, and as an essential DNA-contacting component during DNA translocation. The data also imply that, with the exception of the C18C19A20 bulge, the main role of the 5'/3' helical double-stranded region of pRNA is not for procapsid binding but for binding to gp16.     

Structure of the RNA claw of the DNA packaging motor of bacteriophage ?29

Bacteriophage DNA packaging motors translocate their genomic DNA into viral heads, compacting it to near-crystalline density. The Bacillus subtilis phage ϕ29 has a unique ring of RNA (pRNA) that is an essential component of its motor, serving as a scaffold for the packaging ATPase. Previously, deletion of a three-base bulge (18-CCA-20) in the pRNA A-helix was shown to abolish packaging activity. Here, we solved the structure of this crucial bulge by nuclear magnetic resonance (NMR) using a 27mer RNA fragment containing the bulge (27b). The bulge actually involves five nucleotides (17-UCCA-20 and A100), as U17 and A100 are not base paired as predicted. Mutational analysis showed these newly identified bulge residues are important for DNA packaging. The bulge introduces a 33–35° bend in the helical axis, and inter-helical motion around this bend appears to be restricted. A model of the functional 120b pRNA was generated using a 27b NMR structure and the crystal structure of the 66b prohead-binding domain. Fitting this model into a cryo-EM map generated a pentameric pRNA structure; five helices projecting from the pRNA ring resemble an RNA claw. Biochemical analysis suggested that this shape is important for coordinated motor action required for DNA translocation.     

Boundary of pRNA functional domains and minimum pRNA sequence requirement for specific connector binding and DNA packaging of phage phi29.

Bacteriophage phi29 utilizes a viral-encoded 120-base RNA (pRNA) to accomplish dsDNA packaging into a preformed procapsid. Six pRNAs bind to the procapsid and work sequentially. The pRNA contains two functional domains, one for binding to the DNA translocating connector, and the other for interacting with another component of the DNA packaging machinery during DNA translocation. By UV crosslinking, the pRNA was found to bind to the connector specifically and not to the capsid or scaffolding proteins. When purified connectors were incubated with pRNA, rosette-like connector oligomers were observed. These oligomers were found to contain pRNA. A series of deletion mutants of the pRNA were constructed and their ability to perform various tasks involved in phi29 assembly were assayed. The minimum sizes of the pRNA needed for the following activities have been determined: (1) specific binding to procapsid or to connectors; (2) connector or procapsid binding with full efficiency compared with wild-type pRNA; and (3) genomic DNA packaging. In summary, bases 37-91 (55 nt) comprised the minimum sequence required for specific connector binding, although with lower efficiency; bases 6-113 (105 nt with the additional deletion of two nonessential bases, C109 and A106) comprised the minimum sequence required for full connector binding activity; and bases 1-117 comprised the minimum sequence needed for full DNA packaging activity. These data indicate clearly that the helical region composed of bases 1-6 and 113-117 plays a crucial role in DNA translocation, but is dispensable for connector binding. A model for the role of the pRNA in DNA packaging was also presented.     

Analysis of intermolecular base pair formation of prohead RNA of the phage phi29 DNA packaging motor using NMR spectroscopy

The bacteriophage ø29 DNA packaging motor that assembles on the precursor capsid (prohead) contains an essential 174-nt structural RNA (pRNA) that forms multimers. To determine the structural features of the CE- and D-loops believed to be involved in multimerization of pRNA, 35- and 19-nt RNA molecules containing the CE-loop or the D-loop, respectively, were produced and shown to form a heterodimer in a Mg²⁺-dependent manner, similar to that with full-length pRNA. It has been hypothesized that four intermolecular base pairs are formed between pRNA molecules. Our NMR study of the heterodimer, for the first time, proved directly the existence of two intermolecular Watson–Crick G–C base pairs. The two potential intermolecular A–U base pairs were not observed. In addition, flexibility of the D-loop was found to be important since a Watson–Crick base pair introduced at the base of the D-loop disrupted the formation of the intermolecular G–C hydrogen bonds, and therefore affected heterodimerization. Introduction of this mutation into the biologically active 120-nt pRNA (U80C mutant) resulted in no detectable dimerization at ambient temperature as shown by native gel and sedimentation velocity analyses. Interestingly, this pRNA bound to prohead and packaged DNA as well as the wild-type 120-nt pRNA.     

A viral RNA that binds ATP and contains a motif similar to an ATP-binding aptamer from SELEX

The intriguing process of free energy conversion, ubiquitous in all living organisms, is manifested in ATP binding and hydrolysis. ATPase activity has long been recognized to be a capability limited to proteins. However, the presence of an astonishing variety of unknown RNA species in cells and the finding that RNA has catalytic activity have bred the notion that RNA might not be excluded from the group of ATPases. All DNA-packaging motors of double-stranded DNA phages involve two nonstructural components with certain characteristics typical of ATPases. In bacterial virus phi29, one of these two components is an RNA (pRNA). Here we report that this pRNA is able to bind ATP. A comparison between the chemically selected ATP-binding RNA aptamer and the central region of pRNA reveals similarity in sequence and structure. The replacement of the central region of pRNA with the sequence from ATP-binding RNA aptamer produced chimeric aptRNA that is able to both bind ATP and assemble infectious viruses in the presence of ATP. RNA mutation studies revealed that changing only one base essential for ATP binding caused both ATP binding and viral assembly to cease, suggesting that the ATP binding motif is the vital part of the pRNA that forms a hexamer to drive the phi29 DNA-packaging motor. This is the first demonstration of a natural RNA molecule that binds ATP and the first case to report the presence of a SELEX-derived RNA aptamer in living organisms.     

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.     

The RNA helicase DHX9 establishes nucleolar heterochromatin,and this activity is required for embryonic stem cell differentiation

Long non‐coding RNAs (lncRNAs) have been implicated in the regulation of chromatin conformation and epigenetic patterns. lncRNA expression levels are widely taken as an indicator for functional properties. However, the role of RNA processing in modulating distinct features of the same lncRNA is less understood. The establishment of heterochromatin at rRNA genes depends on the processing of IGS‐rRNA into pRNA, a reaction that is impaired in embryonic stem cells (ESCs) and activated only upon differentiation. The production of mature pRNA is essential since it guides the repressor TIP5 to rRNA genes, and IGS‐rRNA abolishes this process. Through screening for IGS‐rRNA‐binding proteins, we here identify the RNA helicase DHX9 as a regulator of pRNA processing. DHX9 binds to rRNA genes only upon ESC differentiation and its activity guides TIP5 to rRNA genes and establishes heterochromatin. Remarkably, ESCs depleted of DHX9 are unable to differentiate and this phenotype is reverted by the addition of pRNA, whereas providing IGS‐rRNA and pRNA mutants deficient for TIP5 binding are not sufficient. Our results reveal insights into lncRNA biogenesis during development and support a model in which the state of rRNA gene chromatin is part of the regulatory network that controls exit from pluripotency and initiation of differentiation pathways.     

pRNA导入型Ribozyme的设计和体外转录制备

目的:枯草杆菌的包装RNA分子pRNA是新型纳米分子载体,将其同锤头型核酶Ribozyme重组可以构建结构稳定、能进入细胞、主动识别结合和剪切基因RNA的pRNA-Ribozyme.由于目前100 nt以上的RNA分子采用化学合成制备较为困难,实验采用基因重组构建并体外转录制备170 nt的pRNA-Ribozyme....     

Sequential action of six virus-encoded DNA-packaging RNAs during phage phi29 genomic DNA translocation.

A 120-base pRNA encoded by bacteriophage b29 has a novel and essential role in genomic DNA packaging. Six DNA-packaging RNAs (pRNAs) were bound to the sixfold symmetrical portal vertex of procapsids during the DNA translocation process and left the procapsid after the DNA-packaging reaction was completed, suggesting that the pRNA participated in the translocation of genomic DNA into procapsids. To further investigate the mechanism of DNA packaging, it is crucial to determine whether these six pRNA molecules work as an integrated entity or each pRNA acts as a functional individual. If pRNAs work individually, then do they work in sequence with communication or in random order without interaction? Results from compensation and complementation analysis did not support the integrated model. Computation of the probability of combination between wild-type and mutant pRNAs and experimental data of competitive inhibition excluded the random model while favoring the proposal that the six pRNAs functioned sequentially. Sequential action of the pRNA also explains why the pRNA is so sensitive to mutation, since the effect of a pRNA mutation will be amplified by 6 orders of magnitude after six consecutive steps, resulting in the observed complete loss of DNA-packaging activity caused by small alterations. When any one of the six pRNAs was replaced with an inactive one, complete blockage of DNA packaging resulted, strongly supporting the speculation that individual pRNAs, presumably together with other components such as the packaging ATPase gp16, take turns mediating successive steps of packaging. Although the data provided here could not exclude the integrated model completely, there is no evidence so far to argue against the model of sequential action.     

A Three-Helix Junction Is the Interface between Two Functional Domains of Prohead RNA in ?29 DNA Packaging

The double-stranded-DNA bacteriophages employ powerful molecular motors to translocate genomic DNA into preformed capsids during the packaging step in phage assembly. Bacillus subtilis bacteriophage ϕ29 has an oligomeric prohead RNA (pRNA) that is an essential component of its packaging motor. The crystal structure of the pRNA-prohead binding domain suggested that a three-helix junction constitutes both a flexible region and part of a rigid RNA superhelix. Here we define the functional role of the three-helix junction in motor assembly and DNA packaging. Deletion mutagenesis showed that a U-rich region comprising two sides of the junction plays a role in the stable assembly of pRNA to the prohead. The retention of at least two bulged residues in this region was essential for pRNA binding and thereby subsequent DNA packaging. Additional deletions resulted in the loss of the ability of pRNA to multimerize in solution, consistent with the hypothesis that this region provides the flexibility required for pRNA oligomerization and prohead binding. The third side of the junction is part of a large RNA superhelix that spans the motor. The insertion of bases into this feature resulted in a loss of DNA packaging and an impairment of initiation complex assembly. Additionally, cryo-electron microscopy (cryoEM) analysis of third-side insertion mutants showed an increased flexibility of the helix that binds the ATPase, suggesting that the rigidity of the RNA superhelix is necessary for efficient motor assembly and function. These results highlight the critical role of the three-way junction in bridging the prohead binding and ATPase assembly functions of pRNA.