Gp16

The two noncapsid DNA-packaging proteins of various bacteriophages apparently cooperate to link the procapsid to the DNA-packaging substrate, cut monomers of DNA from concatemers, and serve as a DNA-translocation enzyme and/or ATPase. It was found that gp16 of ^29 contains both A-type and B-type consensus ATP-binding sequences, and the predicted secondary structure for ATP binding (30). The A-type sequence of gp16 is "basic-hydro-phobic region-G-X2-G-X-G-K-S-X7 hydrophobic." One of the nonstructural DNA-packaging proteins of all dsDNA phages likely functions as an ATPase (30). This prediction has been supported by subsequent studies involving bacteriophages T4 (150), X (38,152,153), T3 (154), and P22 (155), as well as herpesviruses (88,90) and the vaccinia virus (57).

gp16 is quite hydrophobic, and when overproduced in E. coli, it agglutinates into lumps and loses biological activity. A very simple procedure has been developed to purify gp16 to homogeneity in a soluble, active form, in a single purification step (9). The lumps (inclusion bodies) are collected by differential centrifugation, and the protein, denatured and dissolved with guanidine chloride, is passed through a Sephadex column in the presence of 7.5 M urea. gp16

is eluted as a discrete peak with >90% purity. The purified gp16 is then rena-tured by dialysis against potassium chloride (9). It has been shown that two unique cysteine residues of gp16 are important for biological activity, and, therefore, the reducing agent dithiothreitol is strictly required when guanidine chloride is used for denaturation.

gp16 has been found to bind ATP (19,30,156,157), but the role of gp16 in the ^29 DNA-packaging motor is still a mystery. The hydrophobicity, low solubility, and self-aggregation of ^29 gp16 have long hindered further refinement of the current understanding of its packaging mechanism. Contradictory data regarding ATPase activity, binding location, and the stoichiometry of gp16 have been published (19,30). In the traditional method (30), gp16 was purified in a denatured condition, and active gp16 was obtained by dialysis against 4 ml KCl buffer for 40 min for renaturation, but the renatured gp16 aggregated again within 15 min after renaturation. Recently, it was reported that gp16 was made soluble in the cell by coexpression with groE (157). Indeed, active gp16 was purified through use of this overexpression system. However, although coexpression with groE solved the problem of aggregation within the cell, it could not solve the problem of self-aggregation after purification. PEG and acetone have been used to aid in the purification of gp16 in a homogenous and highly active form (19,259).

Even considering its similarities to other dsDNA viruses regarding DNA, several aspects of the specific structure of ^29 are novel and unique, and it is therefore a particularly promising choice for further study. A 120 nt RNA (discovered in 1987 [98]) is vital to proper functioning of the motor. This pRNA binds ATP (80) and enhances the ATPase activity of gp16 (156). A computer model of the pRNA/connector complex has been reported (158). Notably, pRNA binds the connector and participates actively in DNA translocation (79,159), but it leaves the capsid after DNA packaging is completed (160); ^29-encoded pRNA is not present in the mature virion (98). Similar but distinct pRNAs have been revealed through phylogenetic analyses of other phages; there are few conserved bases, but the observed secondary structures are quite similar (161,162). pRNAs from other phages can never replace ^29 pRNA in its DNA packaging (161).

2.7.1. Two Domains of pRNA

The two functional domains of pRNA are the procapsid-binding domain, located at its central region (159,163-165), bases 23 to 97, and the DNA translocation domain, located at the 5'/3' paired ends (166). A number of different approaches have been employed to confirm this result, including base deletion and mutation (166-169), ribonuclease probing (160,161,163), oligo targeting (170), competition assays to inhibit phage assembly (171-173), ultraviolet crosslinking to portal protein (159), psoralen crosslinking (160), and computer modeling (158). It has been reported that the C18C19A20 bulge of pRNA is solvent exposed when pRNA is bound to procapsid (174) and is critical for DNA translocation (143,162,169) (163). The C18C19A20 bulge might be directly involved in interacting with ATP, gp16, DNA-gp3, or capsid components.

2.7.2. Formation of Dimers, Trimers, and Hexamers pRNA dimer is the building block and dimer ^ tetramer ^ hexamer is the pathway to hexamer assembly. Six individual ^29 pRNAs form a ring through intermolecular base pairing between the right loop (bases 45-48) and the left loop (bases 82-85) (143). Cryo-atomic force microscopy (cryo-AFM) has been used to directly visualize the tertiary structure of pRNA monomers, native and covalently linked dimers, and native trimers. The pRNA monomers fold into a checkmark-shaped structure, the dimers have an elongated shape, and the trimers exhibit a triangular shape. Mg2+ induces appropriate folding of pRNA for dimerization (160,174). pRNA dimers bind to the portal vertex (connector) and serve as the building blocks for hexamer assembly (164,174). The DNA-translocating machine is geared by the formation of a hexameric complex; this suggests that the mechanism employed is similar to that of the consecutive firing of six cylinders of a car engine. Base pairing is demonstrated through an experimental procedure in which pRNAs with mutated loop sequences are constructed. Uppercase and lowercase letters have been used in the literature to indicate the right- and left-hand loops of the pRNA, respectively. Complementary sequences are delineated by the same letter in uppercase and lowercase. For example, in pRNA A-a', right loop A (5'GGAC48) and left loop a' (3'CCUG82) are complementary, whereas in pRNA A-b', the four bases of the right loop A are not complementary to the sequence of the left loop b' (3'UGCG82). Mutant pRNAs with complementary loop sequences (such as pRNA I/i') are active in ^29 DNA packaging, whereas mutants with non-complementary loops (such as pRNA A/b') are inactive (175,176). It was found that pRNAs A-i' and I-a' were inactive in DNA packaging alone, but when A-i' and I-a' were mixed together, DNA-packaging activity was restored. This result is explained by the transcomplementarity of pRNA loops; the right-hand loop A of pRNA A-i' pairs with the left-hand loop a' of pRNA I-a' (175,176).

2.7.3. Stoichiometric Determination of pRNA

As a first step in the determination of pRNA's role in DNA packaging, it is important to conclusively determine how many copies of pRNA are used in each DNA-packaging event. A number of methods have been proven useful in determining the stoichiometry of pRNA and of the other components necessary for motor function (163,172,175,177,178). These various approaches have indicated that six pRNAs are present in the ^29 DNA-translocation motor. Such methods include binomial distribution (172,178) ; comparing slopes of concentration dependence (103,172); and finding the common multiples of 2, 3, and 6 by using sets of two interlocking pRNAs, three interlocking pRNAs, and six interlocking pRNAs (175).

2.7.3.1. Binomial Distribution (172,178)

pRNAs mutated at the 5'/3' paired region retain procapsid-binding capacity but lose DNA-packaging function. When mutant pRNA and wild-type (WT) (Fig. 1) pRNA are mixed at various ratios for in vitro assembly assays (e.g., 30% mutant and 70% WT), the probability of procapsids possessing a certain amount of mutant and a certain amount of WT pRNA can be determined by expanding the binomial as follows:

in which = [Z!/M!(Z - M)!]; Z is the total number of pRNAs per procapsid;

and p and q are the percentage of mutant and WT pRNA, respectively, used in the reaction mixture. For example, if the total number of pRNA per procapsid required for DNA packaging (Z) is 3, then the probability of all combinations of mutant (M) and WT (N) pRNAs on a given procapsid can be determined by expansion of the binomial: (p + q)3 = p3 + 3p2q + 3pq2 + q3 = 100%. That is, in the procapsid population, it is possible to determine the probability of a procapsid possessing either three copies of mutant pRNA, two copies of mutant and one copy of WT, one copy of mutant and two copies of WT, or three copies of WT, represented by p3, 3p2q, 3pq2, and q3, respectively. Suppose that there are 70% mutant and 30% WT pRNA in a reaction mixture. The percentage of procapsids that possess one copy of mutant and two copies of WT would then be 3pq2, i.e., 3(0.7)(0.3)2 = 19%. If only one mutant pRNA per procapsid is sufficient to render the procapsid unable to package DNA, then only those procapsids with three bound WT pRNAs (zero mutant pRNA, q3) would package DNA. The yield of virions from the empirical data was plotted and compared to a series of predicted curves to find a best fit. It was found that the experimental inhibition curve most closely resembled, in both slope and magnitude, the predicted curves in which Z = 5 or 6 (172).

Fig. 1. Structure, domain and location of pRNA on ^29 viral particle. (A), Secondary structure of pRNA(A-b'). A and b' are the left and right loops responsible for intermolecular interaction. The intermolecular binding domain (shaded area) and the reactive DNA translocation domain are marked with bold lines. The four bases in the right and left loops, which are responsible for inter-molecule interactions, are placed in boxes. (B), Power Rangers to depict pRNA hexamer by hand-in-hand interaction. (C), ^29 particle showing the DNA-packaging motor with pRNA hexamer formed via the interaction of pRNA (A-b') and (B-a'). The surrounding pentagon stands for the fivefold symmetrical capsid vertex, viewed as end-on with the virion as a side view. The central region of pRNA binds to the connector and the 5'/3' paired region extends outward (From ref. 162 with permission.)

Fig. 1. Structure, domain and location of pRNA on ^29 viral particle. (A), Secondary structure of pRNA(A-b'). A and b' are the left and right loops responsible for intermolecular interaction. The intermolecular binding domain (shaded area) and the reactive DNA translocation domain are marked with bold lines. The four bases in the right and left loops, which are responsible for inter-molecule interactions, are placed in boxes. (B), Power Rangers to depict pRNA hexamer by hand-in-hand interaction. (C), ^29 particle showing the DNA-packaging motor with pRNA hexamer formed via the interaction of pRNA (A-b') and (B-a'). The surrounding pentagon stands for the fivefold symmetrical capsid vertex, viewed as end-on with the virion as a side view. The central region of pRNA binds to the connector and the 5'/3' paired region extends outward (From ref. 162 with permission.)

2.7.3.2. Slopes of Concentration Dependence (103,172,179)

The dose response curves of in vitro ^29 assembly vs concentration of various assembly components have been used to approximate the stoichiometry of pRNA. ^29 assembly components with known stoichiometry serve as standard controls. In these curves, the larger the stoichiometry of the component, the more dramatic the influence of the dilution factor on the reaction. A slope of one indicates that one copy of the component is involved in the assembly of one virion, as is the case for genomic DNA in ^29 assembly. A slope larger than one indicates multiple-copy involvement. Using this method, the stoichio-metries of DNA-gp3, gp11, gp12, and gp16 have been compared. The result is consistent with other approaches in that the stoichiometry of gpll and gp12 is greater than the stoichiometry of gp16 and pRNA. It is known that the stoichiometry of gpll and gp12 in the ^29 assembly is 12, whereas the stoichiometry of pRNA is between 5 and 6. Interestingly, the curves for gp16 and pRNA do not overlap. Whether such a difference is owing to the possible reusability of pRNA or gpl6 remains to be elucidated.

2.7.3.3. Common Multiples of 3 and 6

This stoichiometric method begins with mixing two inactive mutant pRNAs intermolecularly with complementary loops, and then assaying the activity of such mixtures in DNA packaging (175,176). The predicted secondary structure of the pRNA reveals two loops, known as the left- and right-hand loops (162). Sequences of these two loops (bases 45-48 of the right-hand loop and bases 85-82 of the left-hand loop) are complementary and were originally proposed to form a pseudoknot (165). However, numerous studies have revealed that these two sequences interact intermolecularly, allowing the formation of pRNA multimers. Several pRNAs with mutated left- and right-hand loop sequences have been constructed.

2.7.3.3.1. A Set of Two Interlocking pRNAs (175)

An important finding in pRNA research was that mixing two mutant pRNAs with transcomplementary loops restores full DNA-packaging activity. pRNAs A/b' and B/a' are inactive in packaging when alone, but when they are mixed together, DNA-packaging activity is restored; the right-hand loop A of pRNA A/b' pairs with the left-hand loop a' of pRNA B/a'. Since mixing two inactive pRNAs with interlocking loops—such as when pRNA I-j' and J-i' are mixed in a 1:1 molar ratio—results in the production of infectious virions, the stoichiometry of the pRNA is predicted to be a multiple of two. This, taken together with the results from binomial distribution and serial dilution analyses, strongly indicates that the stoichiometry of pRNA is six.

2.7.3.3.2. A Set of Three Interlocking pRNAs (175)

A similar procedure was used to confirm the formation of trimers through hand-in-hand interaction. Another set of mutants was composed using three pRNAs: A-b', B-c', and C-a'. This set is expected geometrically to form a 3, 6, 9, or 12mer ring that carries each of the three mutants. When tested alone, each individual pRNA exhibited little or no activity. When any two of the three mutants were mixed, again little or no activity was detected. However, when all three pRNAs were mixed in a 1:1:1 ratio, DNA-packaging activity was fully restored. The lack of activity in mixtures of only two mutant pRNAs and the restored activity in mixtures of three mutant pRNAs is to be expected, since the mutations in each RNA are engineered such that only the presence of all three RNAs will produce a closed ring. Since three inactive pRNAs are fully active when mixed together, this suggests that the number of pRNAs in the DNA-packaging complex is a multiple of 3, in addition to being a multiple of 2. Thus, the number of pRNAs required for DNA packaging must be a common multiple of 2 and 3, likely 6 (or 12, but this number has been conclusively excluded by binomial distribution and serial dilution analyses (172), which revealed pRNA's stoichiometry to be between five and six, confirming that the stoichiometry of pRNA is six).

2.7.3.3.3. A Set of Six Interlocking pRNAs (175)

DNA-packaging activity can also be achieved by mixing six mutant pRNAs, each of which is inactive when used alone. Thus, an interlocking hexameric ring is predicted to form by base pairing of the interlocking loops.

2.7.3.3.4. Stoichiometry of Other ^29 Components

The highly sensitive in vitro ^29 assembly system has been utilized to determine the stoichiometry of each of the components needed for assembly (103,104,172,173,178). The log/log plot method incorporates only the functional unit (oligo or complex), not the copy number of the subunits. The procapsid, despite containing (26) copies of scaffolding protein gp7, 235 copies of capsid protein gp8, and 12 copies of the portal protein gp10, is a complex that is regarded as one component. DNA-gp3 is also treated as one component since the DNA and terminal protein gp3 are covalently linked. The log/log plot has revealed that the slope of the log/log concentration-dependent curve for procapsid is one; therefore, one copy of procapsid is needed for the assembly of one virion (103,104,172,173,178). The genomic DNA-gp3 acts in the same way. The stoichiometry for pRNA, gp11, and gp12 has been determined to be 6, 12, and 12, respectively, using log/log slope assay (178), binomial distribution (172,178), common multiples of two and three (175,180), migration rates in gel (181), and electron microscopy (EM) (182,183). Although it has been found that six pRNAs form a hexamer as a vital part of the DNA-translocating motor, the hexamer can be assembled in vitro from individual pRNA molecules or complexes. If the hexamer is assembled from one A-a' or B-b' monomer or six monomers A-b', B-c', C-d', D-e', E-f, F-a' (175), the monomer is regarded as one component and the stoichiometry of the pRNA monomer is six (162). If the hexamer is assembled from purified dimer composed of (I-a')/(A-i'), the dimer is regarded as one component, and the stoichiometry for dimer RNA is three (164,175). If the hexamer is assembled from purified (A-b')/(B-c')/(C-a') trimer, the trimer is regarded as one component and the stoichiometry of tri-mer-RNA is two (162,175). It was found that gp12 formed a dimer before being packaged into a ^29 assembly intermediate (184,185), and each dimeric gp12 is regarded as one component. Therefore, the stoichiometry of gp12 is 12, instead of 24, disregarding the fact that there are 24 copies of gp12 in each ^29 virion. Purified tail protein gp9, expressed in Escherichia coli, has been shown to be active in in vitro ^29 assembly. The absolute copy number of gp9 in one virion has been reported to be nine (181).

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