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.     

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.     

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.     

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.     

Portal-large terminase interactions of the bacteriophage T4 DNA packaging machine implicate a molecular lever mechanism for coupling ATPase to DNA translocation

DNA packaging by double-stranded DNA bacteriophages and herpesviruses is driven by a powerful molecular machine assembled at the portal vertex of the empty prohead. The phage T4 packaging machine consists of three components: dodecameric portal (gp20), pentameric large terminase motor (gp17), and 11- or 12-meric small terminase (gp16). These components dynamically interact and orchestrate a complex series of reactions to produce a DNA-filled head containing one viral genome per head. Here, we analyzed the interactions between the portal and motor proteins using a direct binding assay, mutagenesis, and structural analyses. Our results show that a portal binding site is located in the ATP hydrolysis-controlling subdomain II of gp17. Mutations at key residues of this site lead to temperature-sensitive or null phenotypes. A conserved helix-turn-helix (HLH) that is part of this site interacts with the portal. A recombinant HLH peptide competes with gp17 for portal binding and blocks DNA translocation. The helices apparently provide specificity to capture the cognate prohead, whereas the loop residues communicate the portal interaction to the ATPase center. These observations lead to a hypothesis in which a unique HLH-portal interaction in the symmetrically mismatched complex acts as a lever to position the arginine finger and trigger ATP hydrolysis. Transiently connecting the critical parts of the motor; subdomain I (ATP binding), subdomain II (controlling ATP hydrolysis), and C-domain (DNA movement), the portal-motor interactions might ensure tight coupling between ATP hydrolysis and DNA translocation.     

Cloning, overexpression and purification of the terminase proteins gp16 and gp17 of bacteriophage T4. Construction of a defined in-vitro DNA packaging system using purified terminase proteins

Terminases of double-stranded DNA bacteriophages are required for packaging and generation of terminii in replicated concatemeric DNA molecules. Genetic evidence suggests that these functions in phage T4 are carried out by the products of genes 16 and 17. We cloned these T4 genes into a heat-inducible cI repressor-lambda PL promoter vector system, and overexpressed them in Escherichia coli. We developed an in-vitro DNA packaging system, which, consistent with the genetic data, shows an absolute requirement for the terminase proteins. The overexpressed terminase proteins gp16 and gp17 appear to form a specific complex and an ATP binding site is present in the gp17 molecule. We purified the terminase proteins either as individual gp16 or gp17 proteins, or as a gp16-gp17 complex. The gp16 function of the terminase complex is dispensable for packaging mature DNA, whereas gp17 is essential for packaging DNA under any condition tested. We constructed a defined in-vitro DNA packaging system with the purified terminase proteins, purified proheads and a DNA-free phage completion gene products extract. All the components of this system can be stored at -90 degrees C without loss of packaging activity. The terminase proteins, therefore, may serve as useful reagents for mechanistic studies on DNA packaging, as well as to develop T4 as a packaging-cloning vector.     

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.     

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.     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: oriented and quantized in vitro packaging of DNA protein gp3.

The assembly of phage phi 29 occurs by a single pathway, and the DNA protein (DNA-gp3) of "packaging intermediates" can be obtained after DNase I interruption of in vitro complementation. A broad spectrum of DNA molecules of variable length was isolated from DNase I-treated proheads. Restriction endonuclease EcoRI digestion and electrophoretic analysis of these DNA molecules suggested that DNA-gp3 packaging was oriented with respect to the physical map and was a complex process. Proteinase K-treated exogenous DNA was not packaged. When exogenous DNA-gp3 was predigested with the restriction endonucleases BstEII. EcoRI, HpaI, and HpaII, the left-end fragments, ranging in size from 8 to 0.9 megadaltons, were selectively and efficiently packaged. During in vivo and in vitro assembly, DNA-gp3 is packaged into proheads, the "core-scaffolding" protein gp7 exits from the particles, and the DNA-filled heads assume the angular morphology of phage phi 29. The packaging of a 4.1-megadalton DNA-gp3 left-end fragment (one third of the genome) resulted in the exit of gp7 and the transition to angularity.     

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.     

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.     

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.     

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.     

Characterization of the bacteriophage T3 DNA packaging reaction in vitro in a defined system

The bacteriophage T3 DNA packaging system in vitro defined here is composed of purified proheads and two non-capsid proteins, the products of genes 18 and 19 (gp18 and gp19). In this system, a precursor complex (50 S complex) accumulates in the presence of adenosine 5'-O-(3'-thiotriphosphate) (ATP-gamma-S), a non-hydrolyzable analog of ATP. The 50 S complex is converted to a filled head in the presence of ATP. The conversion of the 50 S complex, formed by preincubation with ATP-gamma-S, to the mature head proceeds in a synchronous manner after the addition of ATP. The lag time for formation of mature heads from the 50 S complex is 1.8, 4.5 and 6.8 minutes at 30, 25 and 20 degrees C, respectively. DNA is translocated into the capsid at a constant rate of 5.7 x 10(3) base-pairs per minute at 20 degrees C. The conversion of the 50 S complex to the mature head exhibits a sigmoidal relationship with respect to the concentration of ATP, the concentration for half-maximal activity being about 20 microM. The transition of the prohead to the expanded capsid occurs at 20 degrees C at one minute 40 seconds after the initiation of DNA translocation, when one-fourth of the genome has been packaged into a prohead. At the same time, the capsid-DNA complex becomes stable to high concentrations of salt. When DNA translocation is interrupted by the addition of ATP-gamma-S, packaged DNA exists at 0 degrees C as well as at 20 degrees C but the exit of DNA stops after one-third of the genome is inside the capsid. After exit, DNA is retranslocated into the expanded capsid by the addition of ATP at a rate of about 5.7 x 10(3) base-pairs per minute at 20 degrees C. The decrease in concentration of ATP interrupts DNA translocation into the capsid but does not induce DNA exit. Interrupted DNA translocation may be reinitiated by the addition of ATP. DNA exit is not induced by the addition of ATP-gamma-S to mature heads or partially filled heads pretreated with DNase.     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: DNA-gp3 intermediate in in vivo and in vitro assembly

The assembly of phage phi 29 occurs by a single pathway, and DNA-protein (DNA-gp3) has been shown to be an intermediate on the assembly pathway by a highly efficient in vitro complementation. At 30 degrees C, about one-half of the viral DNA synthesized was assembled into mature phage, and the absolute plating efficiency of phi 29 approached unity. DNA packaging at 45 degrees C was comparable to that at 30 degrees C, but the burst size was reduced by one-third. When cells infected with mutant ts3(132) at 30 degrees C to permit DNA synthesis were shifted to 45 degrees C before phage assembly, DNA synthesis ceased and no phage were produced. However, a variable amount of DNA packaging occurred. Superinfection by wild-type phage reinitiated ts3(132) DNA synthesis at 45 degrees C, and if native gp3 was covalently linked to this DNA during superinfection replication, it was effectively packaged and assembled. Treatment of the DNA-gp3 complex with trypsin prevented in vitro maturation of phi 29, although substantial DNA packaging occurred. A functional gp3 linked to the 5' termini of phi 29 DNA is a requirement for effective phage assembly in vivo and in vitro.     

Membrane-associated assembly of a phage T4 DNA entrance vertex structure studied with expression vectors

The DNA entrance vertex of the phage head is critical for prohead assembly and DNA packaging. A single structural protein comprises this dodecameric ring substructure of the prohead. Assembly of the phage T4 prohead occurs on the cytoplasmic membrane through a specific attachment at or near the gp20 DNA entrance vertex. An auxiliary head assembly gene product, gp40, was hypothesized to be involved in assembling the gp20 substructure. T4 genes 20, 40 and 20 + 40 were cloned into expression vectors under lambda pL promoter control. The corresponding T4 gene products were synthesized in high yield and were active as judged by their ability to complement the corresponding infecting T4 mutants in vivo. The cloned T4 gene 20 and gene 40 products were inserted into the cytoplasmic membrane as integral membrane proteins; however, gp20 was inserted into the membrane only when gp40 was also synthesized, whereas gp40 was inserted in the presence or absence of gp20. The gp20 insertion required a membrane potential, was not dependent upon the Escherichia coli groE gene, and assumed a defined membrane-spanning conformation, as judged by specific protease fragments protected by the membrane. The inserted gp20 structure could be probed by antibody binding and protein A-gold immunoelectron microscopy. The data suggest that a specific gp20-gp40-membrane insertion structure constitutes the T4 prohead assembly initiation complex.     

Purification and functional characterization of p16, the ATPase of the bacteriophage Φ29 packaging machinery

Bacteriophage Φ29 codes for a protein (p16) that is required for viral DNA packaging both in vivo and in vitro. Co-expression of p16 with the chaperonins GroEL and GroES has allowed its purification in a soluble form. Purified p16 shows a weak ATPase activity that is stimulated by either DNA or RNA, irrespective of the presence of any other viral component. The stimulation of ATPase activity of p16, although induced under packaging conditions, is not dependent of the actual DNA packaging and in this respect the Φ29 enzyme is similar to other viral terminases. Protein p16 competes with DNA and RNA in the interaction with the viral prohead, which occurs through the N-terminal region of the connector protein (p10). In fact, p16 interacts in a nucleotide-dependent fashion with the viral Φ29-encoded RNA (pRNA) involved in DNA packaging, and this binding can be competed with DNA. Our results are consistent with a model for DNA translocation in which p16, bound and organized around the connector, acts as a power stroke to pump the DNA into the prohead, using the hydrolysis of ATP as an energy source.     

DNA packaging and cutting by phage terminases: Control in phage T4 by a synaptic mechanism

Phage DNA packaging occurs by DNA translocation into a prohead. Terminases are enzymes which initiate DNA packaging by cutting the DNA concatemer, and they are closely fitted structurally to the portal vertex of the prohead to form a ‘packasome’. Analysis among a number of phages supports an active role of the terminases in coupling ATP hydrolysis to DNA translocation through the portal. In phage T4 the small terminase subunit promotes a sequence-specific terminase gene amplification within the chromosome. This link between recombination and packaging suggests a DNA synapsis mechanism by the terminase to control packaging initiation, formally homologous to eukaryotic chromosome segregation.     

The functional domains of bacteriophage t4 terminase

The packaging of double-stranded genomic DNA into some viral and all bacteriophage capsids is driven by powerful molecular motors. In bacteriophage T4, the motor consists of the portal protein assembly composed of twelve copies of gene product 20 (gp20, 61 kDa) and an oligomeric terminase complex composed of gp16 (18 kDa) and gp17 (70 kDa). The packaging motor drives the 171-kbp T4 DNA into the capsid utilizing the free energy of ATP hydrolysis. Evidence suggests that gp17 is the key component of the motor; it exhibits ATPase, nuclease, and in vitro DNA-packaging activities. The N- and C-terminal halves of gp17 were expressed and purified to homogeneity and found to have ATPase and nuclease activities, respectively. The N-terminal domain exhibited 2-3-fold higher Kcat values for gp16-stimulated ATPase than the full-length gp17. Neither of the domains, individually or together, exhibited in vitro DNA-packaging activity, suggesting that communication between the domains is essential for DNA packaging. The domains, in particular the C-terminal domain or a mixture of both the N- and C-terminal domains, inhibited in vitro DNA packaging that is catalyzed by full-length gp17. In conjunction with genetic evidence, these data suggest that the domains compete with the full-length gp17 for binding sites on the portal protein. A model for the assembly of the T4 DNA-packaging machine is presented.     

Analysis of capsid portal protein and terminase functional domains: interaction sites required for DNA packaging in bacteriophage T4.

Bacteriophage DNA packaging results from an ATP-driven translocation of concatemeric DNA into the prohead by the phage terminase complexed with the portal vertex dodecamer of the prohead. Functional domains of the bacteriophage T4 terminase and portal gene 20 product (gp20) were determined by mutant analysis and sequence localization within the structural genes. Interaction regions of the portal vertex and large terminase subunit (gp17) were determined by genetic (terminase-portal intergenic suppressor mutations), biochemical (column retention of gp17 and inhibition of in vitro DNA packaging by gp20 peptides), and immunological (co-immunoprecipitation of polymerized gp20 peptide and gp17) studies. The specificity of the interaction was tested by means of a phage T4 HOC (highly antigenicoutercapsid protein) display system in which wild-type, cs20, and scrambled portal peptide sequences were displayed on the HOC protein of phage T4. Binding affinities of these recombinant phages as determined by the retention of these phages by a His-tag immobilized gp17 column, and by co-immunoprecipitation with purified terminase supported the specific nature of the portal protein and terminase interaction sites. In further support of specificity, a gp20 peptide corresponding to a portion of the identified site inhibited packaging whereas the scrambled sequence peptide did not block DNA packaging in vitro.The portal interaction site is localized to 28 residues in the central portion of the linear sequence of gp20 (524 residues). As judged by two pairs of intergenic portal-terminase suppressor mutations, two separate regions of the terminase large subunit gp17 (central and COOH-terminal) interact through hydrophobic contacts at the portal site. Although the terminase apparently interacts with this gp20 portal peptide, polyclonal antibody against the portal peptide appears unable to access it in the native structure, suggesting intimate association of gp20 and gp17 possibly internalizes terminase regions within the portal in the packasome complex. Both similarities and differences are seen in comparison to analogous sites which have been identified in phages T3 and lambda.