Bacteriophage lambda terminase: alterations of the high-affinity ATPase affect viral DNA packaging

DNA packaging by large DNA viruses such as the tailed bacteriophages and the herpesviruses involves DNA translocation into a preformed protein shell, called the prohead. Translocation is driven by an ATP hydrolysis-powered DNA packaging motor. The bacteriophages encode a heterodimeric viral DNA packaging protein, called terminase. The terminases have an ATPase center located in the N terminus of the large subunit implicated in DNA translocation. In previous work with phage lambda, lethal mutations that changed ATP-reactive residues 46 and 84 of gpA, the large terminase subunit, were studied. These mutant enzymes retained the terminase endonuclease and helicase activities, but had severe defects in virion assembly, and lacked the terminase high-affinity ATPase activity. Surprisingly, in the work described here, we found that enzymes with the conservative gpA changes Y46F and Y46A had only mild packaging defects. These mild defects contrast with their profound virion assembly defects. Thus, these mutant enzymes have, in addition to the mild DNA packaging defects, a severe post-DNA packaging defect. In contrast, the gpA K84A enzyme had similar virion assembly and DNA packaging defects. The DNA packaging energy budget, i.e. DNA packaged/ATP hydrolyzed, was unchanged for the mutant enzymes, indicating that DNA translocation is tightly coupled to ATP hydrolysis. A model is proposed in which gpA residues 46 and 84 are important for terminase's high-affinity ATPase activity. Assembly of the translocation complex remodels this ATPase so that residues 46 and 84 are not crucial for the activated translocation ATPase. Changing gpA residues 46 and 84 primarily affects assembly, rather than the activity, of the translocation complex.     

Kinetic characterization of the GTPase activity of phage lambda terminase: evidence for communication between the two "NTPase" catalytic sites of the enzyme.

The terminase enzyme from bacteriophage lambda is responsible for the insertion of viral DNA into the confined space within the capsid. The enzyme is composed of the virally encoded proteins gpA (73.3 kDa) and gpNu1 (20.4 kDa) isolated as a gpA(1).gpNu1(2) holoenzyme complex. Lambda terminase possesses a site-specific nuclease activity, an ATP-dependent DNA strand-separation activity, and an ATPase activity that must work in concert to effect genome packaging. We have previously characterized the ATPase activity of the holoenzyme and have identified catalytic active sites in each enzyme subunit [Tomka and Catalano (1993) Biochemistry 32, 11992-11997; Hwang et al. (1996) Biochemistry 35, 2796-2803]. We have noted that GTP stimulates the ATPase activity of the enzyme, and terminase-mediated GTP hydrolysis has been observed. The studies presented here describe a kinetic analysis of the GTPase activity of lambda terminase. GTP hydrolysis by the enzyme requires divalent metal, is optimal at alkaline pH, and is strongly inhibited by salt. Interestingly, while GTP can bind to the enzyme in the absence of DNA, GTP hydrolysis is strictly dependent on the presence of polynucleotide. Unlike ATP hydrolysis that occurs at both subunits of the holoenzyme, a single catalytic site is observed in the steady-state kinetic analysis of GTPase activity (k(cat) approximately 37 min(-)(1); K(m) approximately 500 microM). Moreover, while GTP stimulates ATP hydrolysis (apparent K(D) approximately 135 microM for GTP binding), all of the adenosine nucleotides examined strongly inhibit the GTPase activity of the enzyme. The data presented here suggest that the two "NTPase" catalytic sites in terminase holoenzyme communicate, and we propose a model describing allosteric interactions between the two sites. The biological significance of this interaction with respect to the assembly and disassembly of the multiple nucleoprotein packaging complexes required for virus assembly is discussed.     

ATPase center of bacteriophage lambda terminase involved in post-cleavage stages of DNA packaging: identification of ATP-interactive amino acids

Terminase is the enzyme that mediates lambda DNA packaging into the viral prohead. The large subunit of terminase, gpA (641 amino acid residues), has a high-affinity ATPase activity (K(m)=5 microM). To directly identify gpA's ATP-interacting amino acids, holoterminase bearing a His(6)-tag at the C terminus of gpA was UV-crosslinked with 8-N(3)-[alpha-(32)P]ATP. Tryptic peptides from the photolabeled terminase were purified by affinity chromatography and reverse-phase HPLC. Two labeled peptides of gpA were identified. Amino acid sequencing failed to show the tyrosine residue of the first peptide, E(43)SAY(46)QEGR(50), or the lysine of the second peptide, V(80)GYSK(84)MLL(87), indicating that Y(46) and K(84) were the 8-N(3)-ATP-modified amino acids. To investigate their roles in lambda DNA packaging, Y(46) was changed to E, A, and F, and K(84) was changed to E and A. Purified His(6)-tagged terminases with changes at residues 46 and 84 lacked the gpA high-affinity ATPase activity, though the cos cleavage and cohesive end separation activities were near to those of the wild-type enzyme. In virion assembly reactions using virion DNA as a packaging substrate, the mutant terminases showed severe defects. In summary, the results indicate that Y(46) and K(84) are part of the high-affinity ATPase center of gpA, and show that this ATPase activity is involved in the post-cos cleavage stages of lambda DNA packaging.     

Defining the ATPase center of bacteriophage T4 DNA packaging machine: requirement for a catalytic glutamate residue in the large terminase protein gp17

Double-stranded DNA packaging in icosahedral bacteriophages is driven by an ATPase-coupled packaging machine constituted by the portal protein and two non-structural packaging/terminase proteins assembled at the unique portal vertex of the empty viral capsid. Recent studies show that the N-terminal ATPase site of bacteriophage T4 large terminase protein gp17 is critically required for DNA packaging. It is likely that this is the DNA translocating ATPase that powers directional translocation of DNA into the viral capsid. Defining this ATPase center is therefore fundamentally important to understand the mechanism of ATP-driven DNA translocation in viruses. Using combinatorial mutagenesis and biochemical approaches, we have defined the catalytic carboxylate residue that is required for ATP hydrolysis. Although the original catalytic carboxylate hypothesis suggested the presence of a catalytic glutamate between the Walker A (SRQLGKT(161-167)) and Walker B (MIYID(251-255)) motifs, none of the four candidate glutamic acid residues, E198, E208, E220 and E227, is required for function. However, the E256 residue that is immediately adjacent to the putative Walker B aspartic acid residue (D255) exhibited a phenotypic pattern that is consistent with the catalytic carboxylate function. None of the amino acid substitutions, including the highly conservative D and Q, was tolerated. Biochemical analyses showed that the purified E256V, D, and Q mutant gp17s exhibited a complete loss of gp16-stimulated ATPase activity and in vitro DNA packaging activity, whereas their ATP binding and DNA cleavage functions remained intact. The data suggest that the E256 mutants are trapped in an ATP-bound conformation and are unable to catalyze the ATP hydrolysis-transduction cycle that powers DNA translocation. Thus, this study for the first time identified and characterized a catalytic glutamate residue that is involved in the energy transduction mechanism of a viral DNA packaging machine.     

The nuclease domain of the SPP1 packaging motor coordinates DNA cleavage and encapsidation

The large terminase subunit is a central component of the genome packaging motor from tailed bacteriophages and herpes viruses. This two-domain enzyme has an N-terminal ATPase activity that fuels DNA translocation during packaging and a C-terminal nuclease activity required for initiation and termination of the packaging cycle. Here, we report that bacteriophage SPP1 large terminase (gp2) is a metal-dependent nuclease whose stability and activity are strongly and preferentially enhanced by Mn²⁺ ions. Mutation of conserved residues that coordinate Mn²⁺ ions in the nuclease catalytic site affect the metal-induced gp2 stabilization and impair both gp2-specific cleavage at the packaging initiation site pac and unspecific nuclease activity. Several of these mutations block also DNA encapsidation without affecting ATP hydrolysis or gp2 C-terminus binding to the procapsid portal vertex. The data are consistent with a mechanism in which the nuclease domain bound to the portal switches between nuclease activity and a coordinated action with the ATPase domain for DNA translocation. This switch of activities of the nuclease domain is critical to achieve the viral chromosome packaging cycle.     

Assembly of bacteriophage lambda terminase into a viral DNA maturation and packaging machine

Terminase enzymes are common to complex double-stranded DNA viruses and function to package viral DNA into the capsid. We recently demonstrated that the bacteriophage lambda terminase gpA and gpNu1 proteins assemble into a stable heterotrimer with a molar ratio gpA1/gpNu1(2). This terminase protomer possesses DNA maturation and packaging activities that are dependent on the E. coli integration host factor protein (IHF). Here, we show that the protomer further assembles into a homogeneous tetramer of protomers of composition (gpA1/gpNu1(2))4. Electron microscopy shows that the tetramer forms a ring structure large enough to encircle duplex DNA. In contrast to the heterotrimer, the ring tetramer can mature and package viral DNA in the absence of IHF. We propose that IHF induced bending of viral DNA facilitates the assembly of four terminase protomers into a ring tetramer that represents the catalytically competent DNA maturation and packaging complex in vivo. This work provides, for the first time, insight into the functional assembly state of a viral DNA packaging motor.     

The bacteriophage lambda terminase. Partial purification and preliminary characterization of properties

The maturation of bacteriophage lambda DNA and its packaging into preformed heads to produce infectious phage is under the control of the two leftmost genes on the lambda chromosome, i.e., Nu1 and A. Based on its ability to complement lambda A- phage-infected cell extracts for packaging of lambda DNA in vitro, a single protein, designated terminase (ter) has been extensively purified using adsorption, ion exchange, and affinity column chromatography. The final preparation represents an approximately 60,000-fold purification over the activity found in crude extracts and is about 30 to 80% homogeneous as judged by visualizing the protein after electrophoresis in sodium dodecyl sulfate-polyacrylamide gel. In addition to packaging, terminase can also catalyze the endonucleolytic cleavage of lambda cohesive-end site DNA; both of these reactions require ATP. In some preparations, certain terminase fractions of extreme purity require protein factors present in extracts of uninfected Escherichia coli in order to catalyze the cohesive-end site cleavage reaction. On ion exchange columns purified terminase co-chromatographs with a DNA-dependent ATPase activity, hydrolyzing ATP to ADP and Pi in the presence of any of several types of DNA tested including those of non-lambda origin. The molecular weight of the native enzyme is 117,000 and appears to be a hetero-oligomer composed of 2 nonidentical subunits. The most likely composition of terminase is one gpA (gene product of A), Mr = 74,000 and two gpNu1, Mr = 21,000.     

The ATPase domain of the large terminase protein, gp17, from bacteriophage T4 binds DNA: implications to the DNA packaging mechanism

Translocation of double-stranded DNA into a preformed capsid by tailed bacteriophages is driven by powerful motors assembled at the special portal vertex. The motor is thought to drive processive cycles of DNA binding, movement, and release to package the viral genome. In phage T4, there is evidence that the large terminase protein, gene product 17 (gp17), assembles into a multisubunit motor and translocates DNA by an inchworm mechanism. gp17 consists of two domains; an N-terminal ATPase domain (amino acids 1-360) that powers translocation of DNA, and a C-terminal nuclease domain (amino acids 361-610) that cuts concatemeric DNA to generate a headful-size viral genome. While the functional motifs of ATPase and nuclease have been well defined and the ATPase atomic structure has been solved, the DNA binding motif(s) responsible for viral DNA recognition, cutting, and translocation are unknown. Here we report the first evidence for the presence of a double-stranded DNA binding activity in the gp17 ATPase domain. Binding to DNA is sensitive to Mg²⁺ and salt, but not the type of DNA used. DNA fragments as short as 20 bp can bind to the ATPase but preferential binding was observed to DNA greater than 1 kb. A high molecular weight ATPase-DNA complex was isolated by gel filtration, suggesting oligomerization of ATPase following DNA interaction. DNA binding was not observed with the full-length gp17, or the C-terminal nuclease domain. The small terminase protein, gp16, inhibited DNA binding, which was further accentuated by ATP. The presence of a DNA binding site in the ATPase domain and its binding properties implicate a role in the DNA packaging mechanism.     

Nucleotides regulate the conformational state of the small terminase subunit from bacteriophage lambda: implications for the assembly of a viral genome-packaging motor

Terminase enzymes are responsible for "packaging" of viral DNA into a preformed procapsid. Bacteriophage lambda terminase is composed of two subunits, gpA and gpNu1, in a gpA(1).gpNu1(2) holoenzyme complex. The larger gpA subunit is responsible for preparation of viral DNA for packaging, and is central to the packaging motor complex. The smaller gpNu1 subunit is required for site-specific assembly of the packaging motor on viral DNA. Terminase assembly at the packaging initiation site is regulated by ATP binding and hydrolysis at the gpNu1 subunit. Characterization of the catalytic and structural interactions between the DNA and nucleotide binding sites of gpNu1 is thus central to our understanding of the packaging motor at the molecular level. The high-resolution structure of the DNA binding domain of gpNu1 (gpNu1-DBD) was recently determined in our lab [de Beer, T., et al. (2002) Mol. Cell 9, 981-991]. The structure reveals the presence of a winged-helix-turn-helix DNA binding motif, but the location of the ATPase catalytic site in gpNu1 remains unknown. In this work, nucleotide binding to the gpNu1-DBD was probed using acrylamide fluorescence quenching and fluorescence-monitored ligand binding studies. The data indicate that the minimal DBD dimer binds both ATP and ADP at two equivalent but highly cooperative binding sites. The data further suggest that ATP and ADP induce distinct conformations of the dimer but do not affect DNA binding affinity. The implications of these results with respect to the assembly and function of a terminase DNA-packaging motor are discussed.     

Self-association properties of the bacteriophage lambda terminase holoenzyme: implications for the DNA packaging motor

Terminases are enzymes common to complex double-stranded DNA viruses and are required for packaging of viral DNA into a protective capsid. Bacteriophage lambda terminase holoenzyme is a hetero-oligomer composed of the A and Nu1 lambda gene products; however, the self-association properties of the holoenzyme have not been investigated systematically. Here, we report the results of sedimentation velocity, sedimentation equilibrium, and gel-filtration experiments studying the self-association properties of the holoenzyme. We find that purified, recombinant lambda terminase forms a homogeneous, heterotrimeric structure, consisting of one gpA molecule associated with two gpNu1 molecules (114.2 kDa). We further show that lambda terminase adopts a heterogeneous mixture of higher-order structures, with an average molecular mass of 528(+/-34) kDa. Both the heterotrimer and the higher-order species possess site-specific cos cleavage activity, as well as DNA packaging activity; however, the heterotrimer is dependent upon Escherichia coli integration host factor (IHF) for these activities. Furthermore, the ATPase activity of the higher-order species is approximately 1000-fold greater than that of the heterotrimer. These data suggest that IHF bending of the duplex at the cos site in viral DNA promotes the assembly of the heterotrimer into a biologically active, higher-order packaging motor. We propose that a single, higher-order hetero-oligomer of gpA and gpNu1 functions throughout lambda development.     

Energy-independent helicase activity of a viral genome packaging motor

The assembly of complex double-stranded DNA viruses includes a genome packaging step where viral DNA is translocated into the confines of a preformed procapsid shell. In most cases, the preferred packaging substrate is a linear concatemer of viral genomes linked head-to-tail. Viral terminase enzymes are responsible for both excision of an individual genome from the concatemer (DNA maturation) and translocation of the duplex into the capsid (DNA packaging). Bacteriophage λ terminase site-specifically nicks viral DNA at the cos site in a concatemer and then physically separates the nicked, annealed strands to mature the genome in preparation for packaging. Here we present biochemical studies on the so-called helicase activity of λ terminase. Previous studies reported that ATP is required for strand separation, and it has been presumed that ATP hydrolysis is required to drive the reaction. We show that ADP and nonhydrolyzable ATP analogues also support strand separation at low (micromolar) concentrations. In addition, the Escherichia coli integration host factor protein (IHF) strongly stimulates the reaction in a nucleotide-independent manner. Finally, we show that elevated concentrations of nucleotide inhibit both ATP- and IHF-stimulated strand separation by λ terminase. We present a model where nucleotide and IHF interact with the large terminase subunit and viral DNA, respectively, to engender a site-specifically bound, catalytically competent genome maturation complex. In contrast, binding of nucleotide to the low-affinity ATP binding site in the small terminase subunit mediates a conformational switch that down-regulates maturation activities and activates the DNA packaging activity of the enzyme. This affords a motor complex that binds tightly, but nonspecifically, to DNA as it translocates the duplex into the capsid shell. These studies have yielded mechanistic insight into the assembly of the maturation complex on viral DNA and its transition to a mobile packaging motor that may be common to all of the complex double-stranded DNA viruses.     

A minimal kinetic model for a viral DNA packaging machine

Terminase enzymes are common to both eukaryotic and prokaryotic double-stranded DNA viruses. These enzymes possess ATPase and nuclease activities that work in concert to "package" a viral genome into an empty procapsid, and it is likely that terminase enzymes from disparate viruses utilize a common packaging mechanism. Bacteriophage lambda terminase possesses a site-specific nuclease activity, a so-called helicase activity, a DNA translocase activity, and multiple ATPase catalytic sites that function to package viral DNA. Allosteric interactions between the multiple catalytic sites have been reported. This study probes these catalytic interactions using enzyme kinetic, photoaffinity labeling, and vanadate inhibition studies. The ensemble of data forms the basis for a minimal kinetic model for lambda terminase. The model incorporates an ADP-driven conformational reorganization of the terminase subunits assembled on viral DNA, which is central to the activation of a catalytically competent packaging machine. The proposed model provides a unifying mechanism for allosteric interaction between the multiple catalytic sites of the holoenzyme and explains much of the kinetic data in the literature. Given that similar packaging mechanisms have been proposed for viruses as dissimilar as lambda and the herpes viruses, the model may find general utility in our global understanding of the enzymology of virus assembly.     

The terminase of bacteriophage lambda. Functional domains for cosB binding and multimer assembly

Terminase is a protein complex involved in lambda DNA packaging. The subunits of terminase, gpNul and gpA, are the products of genes Nul and A. The actions of terminase include DNA binding, prohead binding and DNA nicking. Phage 21 is a lambdoid phage that also makes a terminase, encoded by genes 1 and 2. The terminases of 21 and lambda are not interchangeable. This specificity involves two actions of terminase; DNA binding and prohead binding. In addition, the subunits of lambda terminase will not form functional multimers with the subunits of 21 terminase. lambda-21 hybrid phages can be produced as a result of recombination. We describe here lambda-21 hybrid phages that have hybrid terminase genes. The packaging specificities of the hybrids and the structure of their genes were compared in order to identify functional domains of terminase. The packaging specificities were determined in vivo by complementation tests and helper packaging experiments. Restriction enzyme site mapping and sequencing located the sites at which recombination occurred to produce the hybrid phages. lambda-21 hybrid 51 carries the lambda A gene, and a hybrid 1/Nul gene. The crossover that produced this phage occurred near the middle of the 1 and Nul genes. The amino-terminal portion of the hybrid protein is homologous to gp1 and the carboxy-terminal portion is homologous to gpNul. It binds to 21 DNA and forms functional multimers with gpA, providing evidence that the amino-terminal portion of gpNul is involved in DNA binding and the carboxy-terminal portion of gpNul is involved in the interaction with gpA. lambda-21 hybrid 54 has a hybrid 2/A gene. The amino terminus of the hybrid protein of lambda-21 hybrid 54 is homologous with gp2. This protein forms functional multimers only with gp1, providing evidence that the amino terminus of gpA is involved in the interaction with gpNul. These studies identify three functional domains of terminase.     

Evidence that a phage T4 DNA packaging enzyme is a processed form of the major capsid gene product

A phage T4 DNA packaging enzyme appears to arise as a processed form of the major T4 capsid structural protein gp23. The enzyme activity and antigen are missing from all head gene mutants that block the morphogenetic proteolytic processing reactions of the head proteins in vivo. The enzyme antigen can be formed in vitro by T4 (gp21) specific processing of gp23 containing extracts. Enzyme antigen is found in active processed proheads but not in full heads. The enzyme and the major capsid protein show immunological cross-reactivity, produce common peptides upon proteolysis, and share an assembly-conformation-dependent ATP binding site. The packaging enzyme and the mature capsid protein (gp23*) both appear to arise from processing of gp23, the former as a minor product of a specific gp23 structure in the prohead, acting in DNA packaging as a DNA-dependent ATPase, and a headful-dependent terminase.     

The DNA translocating ATPase of bacteriophage T4 packaging motor

In double-stranded DNA bacteriophages the viral DNA is translocated into an empty prohead shell by a powerful ATP-driven motor assembled at the unique portal vertex. Terminases consisting of two to three packaging-related ATPase sites are central to the packaging mechanism. But the nature of the key translocating ATPase, stoichiometry of packaging motor, and basic mechanism of DNA encapsidation are poorly understood. A defined phage T4 packaging system consisting of only two components, proheads and large terminase protein (gp17; 70 kDa), is constructed. Using the large expanded prohead, this system packages any linear double-stranded DNA, including the 171 kb T4 DNA. The small terminase protein, gp16 (18 kDa), is not only not required but also strongly inhibitory. An ATPase activity is stimulated when proheads, gp17, and DNA are actively engaged in the DNA packaging mode. No packaging ATPase was stimulated by the N-terminal gp17-ATPase mutants, K166G (Walker A), D255E (Walker B), E256Q (catalytic carboxylate), D255E-E256D and D255E-E256Q (Walker B and catalytic carboxylate), nor could these sponsor DNA encapsidation. Experiments with the two gp17 domains, N-terminal ATPase domain and C-terminal nuclease domain, suggest that terminase association with the prohead portal and communication between the domains are essential for ATPase stimulation. These data for the first time established an energetic linkage between packaging stimulation of N-terminal ATPase and DNA translocation. A core pathway for the assembly of functional DNA translocating motor is proposed. Since the catalytic motifs of the N-terminal ATPase are highly conserved among >200 large terminase sequences analyzed, these may represent common themes in phage and herpes viral DNA translocation.     

Cloning, expression, and biochemical characterization of hexahistidine-tagged terminase proteins.

The terminase enzyme from bacteriophage lambda is composed of two viral proteins (gpA, 73.2 kDa; gpNu1, 20.4 kDa) and is responsible for packaging viral DNA into the confines of an empty procapsid. We are interested in the genetic, biochemical, and biophysical properties of DNA packaging in phage lambda and, in particular, the nucleoprotein complexes involved in these processes. These studies require the routine purification of large quantities of wild-type and mutant proteins in order to probe the molecular mechanism of DNA packaging. Toward this end, we have constructed a hexahistidine (hexa-His)-tagged terminase holoenzyme as well as hexa-His-tagged gpNu1 and gpA subunits. We present a simple, one-step purification scheme for the purification of large quantities of the holoenzyme and the individual subunits directly from the crude cell lysate. Importantly, we have developed a method to purify the highly insoluble gpNu1 subunit from inclusion bodies in a single step. Hexa-His terminase holoenzyme is functional in vivo and possesses steady-state and single-turnover ATPase activity that is indistinguishable from wild-type enzyme. The nuclease activity of the modified holoenzyme is near wild type, but the reaction exhibits a greater dependence on Escherichia coli integration host factor, a result that is mirrored in vivo. These results suggest that the hexa-His-tagged holoenzyme possesses a mild DNA-binding defect that is masked, at least in part, by integration host factor. The mild defect in hexa-His terminase holoenzyme is more significant in the isolated gpA-hexa-His subunit that does not appear to bind DNA. Moreover, whereas the hexa-His-tagged gpNu1 subunit may be reconstituted into a holoenzyme complex with wild-type catalytic activities, gpA-hexa-His is impaired in its interactions with the gpNu1 subunit of the enzyme. The results reported here underscore that a complete biochemical characterization of the effects of purification tags on enzyme function must be performed prior to their use in mechanistic studies.     

A functional domain of bacteriophage lambda terminase for prohead binding

Terminase is a multifunctional protein complex involved in DNA packaging during bacteriophage lambda assembly. Terminase is made of gpNul and gpA, the products of the phage lambda Nu1 and A genes. Early during DNA packaging terminase binds to lambda DNA to form a complex called complex I. Terminase is required for the binding of proheads by complex I to form a DNA: terminase: prohead complex known as complex II. Terminase remains associated with the DNA during encapsidation. The other known role for terminase in packaging is the production of staggered nicks in the DNA thereby generating the cohesive ends. Lambdoid phage 21 has cohesive ends identical to those of lambda. The head genes of lambda and 21 show partial sequence homology and are analogous in structure, function and position. The terminases of lambda and 21 are not interchangeable. At least two actions of terminase are involved in this specificity: (1) DNA binding; (2) prohead binding. The 1 and 2 genes at the left end of the 21 chromosome were identified as coding for the 21 terminase. gp1 and gp2 are analogous to gpNu1 and gpA, respectively. We have isolated a phage, lambda-21 hybrid 33, which is the product of a crossover between lambda and 21 within the terminase genes. Lambda-21 hybrid 33 DNA and terminase have phage 21 packaging specificity, as determined by complementation and helper packaging studies. The terminase of lambda-21 hybrid 33 requires lambda proheads for packaging. We have determined the position at which the crossover between lambda DNA and 21 DNA occurred to produce the hybrid phage. Lambda-21 hybrid 33 carries the phage 21 1 gene and a hybrid phage 2/A gene. Sequencing of lambda-21 hybrid 33 DNA shows that it encodes a protein that is homologous at the carboxy terminus with the 38 amino acids of the carboxy terminus of lambda gpA; the remainder of the protein is homologous to gp2. The results of these studies define a specificity domain for prohead binding at the carboxy terminus of gpA.     

Genetic analysis of mutations affecting terminase, the bacteriophage lambda DNA packaging enzyme, that suppress mutations in cosB, the terminase binding site.

Terminase, the DNA packaging enzyme of phage lambda, binds to lambda DNA at a site called cosB, and introduces staggered nicks at an adjacent site, cosN, to generate the cohesive ends of virion lambda DNA molecules. Terminase also is involved in separation of the cohesive ends and in binding the prohead, the empty protein shell into which lambda DNA is packaged. Terminase is a DNA-dependent ATPase, and both subunits, gpNu1 and gpA, have ATPase activity. cosB contains a series of gpNu1 binding sites, R3, R2 and R1; between R3 and R2 is a binding site, I1, for integration host factor (IHF), the Escherichia coli DNA bending protein. In this work, a series of mutations in Nu1 have been isolated as suppressors of cosB mutations. One of the Nu1 mutations is identical to the previously described Nu1ms1/ohm1 mutation predicted to cause the change L40F in the 181 amino acid-long gpNu1. Three other Nu1 missense mutations, the Nu1ms2 (L40I), ms3 (Q97K) and ms4 (A92G) mutations, have been isolated; the relative strengths of suppression of cosB mutations by the Nu1ms mutations are: ms1 > ms2 > ms3 > ms4. The Nu1 missense mutations all affect amino acid residues that lie outside of the putative helix-turn-helix DNA binding motif of gpNu1. The Nu1ms1 and Nu1ms2 mutations alter an amino acid residue (L40) that lies directly between two segments of gpNu1 proposed to be involved in ATP binding and hydrolysis; thus these mutations are likely to alter the gpNu1 ATP-binding site. The Nu1ms3 and Nu1ms4 mutations both affect amino acid residues in the central region of gpNu1 that is predicted to form a hydrophilic alpha-helix. To explain how the Nu1ms mutations suppress cosB defects, models involving alterations of the DNA binding and/or catalytic properties of terminase are considered. The results also indicate that terminase occupancy of a single gpNu1 binding site (R3) is necessary and sufficient for the efficient initiation of DNA packaging; the Nu1ms1, ms2 and ms3 mutations permit IHF-independent plaque formation by a phage lacking R2 and R1.     

Biochemical characterization of an ATPase activity associated with the large packaging subunit gp17 from bacteriophage T4

Double-stranded DNA-packaging in icosahedral bacteriophages is believed to be driven by a packaging "machine" constituted by the portal protein and the two packaging/terminase proteins assembled at the unique portal vertex of the empty prohead shell. Although ATP hydrolysis is evidently the principal driving force, which component of the packaging machinery functions as the translocating ATPase has not been elucidated. Evidence suggests that the large packaging subunit is a strong candidate for the translocating ATPase. We have constructed new phage T4 terminase recombinants under the control of phage T7 promoter and overexpressed the packaging/terminase proteins gp16 and gp17 in various configurations. The hexahistidine-tagged-packaging proteins were purified to near homogeneity by Ni(2+)-agarose chromatography and were shown to be highly active for packaging DNA in vitro. The large packaging subunit gp17 but not the small subunit gp16 exhibited an ATPase activity. Although gp16 lacked ATPase activity, it enhanced the gp17-associated ATPase activity by >50-fold. The gp16 enhancement was specific and was due to an increased catalytic rate for ATP hydrolysis. A phosphorylated gp17 was demonstrated under conditions of low catalytic rates but not under high catalytic rates in the presence of gp16. The data are consistent with the hypothesis that a weak ATPase is transformed into a translocating ATPase of high catalytic capacity after assembly of the packaging machine.