Gene 19 of bacteriophage T7. Overexpression, purification, and characterization of its product

Gene 18 and 19 proteins of bacteriophage T7 are essential for DNA maturation and packaging. The phage capsid is the site of both maturation and packaging of T7 DNA. Both gene 18 and 19 proteins bind to capsid intermediates during DNA packaging but are not found in mature virions, suggesting that they play a direct role in the enzymatic mechanisms of DNA maturation and packaging. As part of an effort to reconstitute T7 DNA maturation and packaging with purified components, we have cloned and overexpressed T7 gene 19 in Escherichia coli. Gene 19 has been inserted downstream from the bacteriophage PL promoter controlled by the temperature-sensitive lambda repressor encoded by c1857. Upon thermal induction, most of the overproduced gene 19 protein is insoluble and inactive. However, by attenuation of the expression of gene 19 from the PL promoter, significant levels of soluble and active gene 19 protein are produced. Soluble gene 19 protein can be monitored by its ability to complement extracts of T7-infected cells for packaging of exogenous DNA. We have used this assay to monitor the activity of gene 19 protein during purification. The native protein is a monomer of molecular weight 66,000. We have also tested for the formation of a stable complex between gene 18 and 19 proteins. Coproduction of gene 18 and 19 proteins has no effect on either the solubility or activity of gene 19 protein, despite the fact that gene 18 protein is produced at at least 10-fold greater rates. Furthermore, we find no evidence for any interaction between soluble gene 18 and 19 proteins in extracts or between the purified proteins.     

Processing of concatemers of bacteriophage T7 DNA in vitro

The T7 chromosome is a double-stranded linear DNA molecule flanked by direct terminal repeats or so-called terminal redundancies. Late in infection bacteriophage T7 DNA accumulates in the form of concatemers, molecules that are comprised of T7 chromosomes joined in a head to tail arrangement through shared terminal redundancies. To elucidate the molecular mechanisms of concatemer processing, we have developed extracts that process concatemeric DNA. The in vitro system consists of an extract of phage T7-infected cells that provides all T7 gene products and minimal levels of endogenous concatemeric DNA. Processing is analyzed using a linear 32P-labeled substrate containing the concatemeric joint. T7 gene products required for in vitro processing can be divided into two groups; one group is essential for concatemer processing, and the other is required for the production of full length left-hand ends. The products of genes 8 (prohead protein), 9 (scaffolding protein), and 19 (DNA maturation) along with gene 18 protein are essential, indicating that capsids are required for processing. In extracts lacking one or more of the products of genes 2 (Escherichia coli RNA polymerase inhibitor), 5 (DNA polymerase), and 6 (exonuclease), full length right-hand ends are produced. However, the left-hand ends produced are truncated, lacking at least 160 base pairs, the length of the terminal redundancy. Gene 3 endonuclease, required for concatemer processing in vivo, is not required in this system. Both the full length left- and right-hand ends produced by the processing reaction are protected from DNase I digestion, suggesting that processing of the concatemeric joint substrate is accompanied by packaging.     

Bacteriophage T7 DNA packaging. III. A "hairpin" end formed on T7 concatemers may be an intermediate in the processing reaction

An unusual left end (M-end) has been identified on bacteriophage T7 DNA isolated from T7-infected cells. This end has a "hairpin" structure and is formed at a short inverted repeat sequence centered around nucleotide 39,587 of T7, 190 base-pairs to the left of the site where a mature left end is formed on the T7 concatemer. We do not detect the companion right end that would be formed if the M-end is produced by a double-stranded cut on the T7 concatemer. This suggests that the hairpin left end may be generated from a single-stranded cut in the DNA that is used to prime rightward DNA synthesis. The formation of M-end does not require the products of T7 genes 10, 18 or 19, proteins that are essential for the formation of mature T7 ends. During infection with a T7 gene 3 (endonuclease) mutant, phage DNA synthesis is reduced and the concatemers are not processed into unit length DNA molecules, but both M-end and the mature right end are formed on the concatemer DNA. These two ends are also found associated with the large, rapidly sedimenting concatemers formed during a normal T7 infection while the mature left end is present only on unit length T7 DNA molecules. We propose that DNA replication primed from the hairpin end produced by a nick in the inverted repeat sequence provides a mechanism to duplicate the terminal repeat before DNA packaging. Packaging is initiated with the formation of a mature right end on the branched concatemer and, as the phage head is filled, the T7 gene 3 endonuclease may be required to trim the replication forks from the DNA. Concatemer processing is completed by the removal of the 190 base-pair hairpin end to produce the mature left end.     

Single-event analysis of the packaging of bacteriophage T7 DNA concatemers in vitro.

Bacteriophage T7 packages its double-stranded DNA genome in a preformed protein capsid (procapsid). The DNA substrate for packaging is a head-to-tail multimer (concatemer) of the mature 40-kilobase pair genome. Mature genomes are cleaved from the concatemer during packaging. In the present study, fluorescence microscopy is used to observe T7 concatemeric DNA packaging at the level of a single (microscopic) event. Metabolism-dependent cleavage to form several fragments is observed when T7 concatemers are incubated in an extract of T7-infected Escherichia coli (in vitro). The following observations indicate that the fragment-producing metabolic event is DNA packaging: 1) most fragments have the hydrodynamic radius (R(H)) of bacteriophage particles (+/-3%) when R(H) is determined by analysis of Brownian motion; 2) the fragments also have the fluorescence intensity (I) of bacteriophage particles (+/-6%); 3) as a fragment forms, a progressive decrease occurs in both R(H) and I. The decrease in I follows a pattern expected for intracapsid steric restriction of 4',6-diamidino-2-phenylindole (DAPI) binding to packaged DNA. The observed in vitro packaging of a concatemer's genomes always occurs in a synchronized cluster. Therefore, the following hypothesis is proposed: the observed packaging of concatemer-associated T7 genomes is cooperative.     

Bacteriophage P1 genes involved in the recognition and cleavage of the phage packaging site (pac).

The packaging of bacteriophage P1 DNA is initiated by cleavage of the viral DNA at a specific site, designated pac. The proteins necessary for that cleavage, and the genes that encode those proteins, are described in this report. By sequencing wild-type P1 DNA and DNA derived from various P1 amber mutants that are deficient in pac cleavage, two distinct genes, referred to as pacA and pacB, were identified. These genes appear to be coordinately transcribed with an upstream P1 gene that encodes a regulator of late P1 gene expression (gene 10). pacA is located upstream from pacB and contains the 161 base-pair pac cleavage site. The predicted sizes of the PacA and PacB proteins are 45 kDa and 56 kDa, respectively. These proteins have been identified on SDS-polyacrylamide gels using extracts derived from Escherichia coli cells that express these genes under the control of a bacteriophage T7 promoter. Extracts prepared from cells expressing both PacA and PacB are proficient for site-specific cleavage of the P1 packaging site, whereas those lacking either protein are not. However, the two defective extracts can complement each other to restore functional pac cleavage activity. Thus, PacA and PacB are two essential bacteriophage proteins required for recognition and cleavage of the P1 packaging site. PacB extracts also contain a second P1 protein that is encoded within the pacB gene. We have identified this protein on SDS-polyacrylamide gels and have shown that it is translated in the same reading frame as is PacB. Its role, if any, in pac cleavage is yet to be determined.     

The structure and maturation of intermediates in bacteriophage T7 DNA replication

Concatemeric DNA from T7-infected cells consists of phage genomes in a linear head-to-tail arrangement. Adjacent genomes within a concatemer overlap for the length of the terminal repetition. Fast-sedimenting T7 DNA contains single-stranded regions at roughly unit-lentth intervals but these interruptions are heterogeneously distributed and do not occur at the genetic termini. Mutations in either bacteriophage genes 9, 18, or 19 (required for DNA maturation and packaging) lead to the synthesis and persistence of DNA with fewer interruptions than normal.     

Purification and characterization of the gene 1.2 protein of bacteriophage T7

Gene 1.2 of bacteriophage T7, located near the primary origin of DNA replication at position 15.37 on the T7 chromosome, encodes a 10,059-dalton protein that is essential for growth on Escherichia coli optA1 strains (Saito, H., and Richardson, C. C. (1981) J. Virol. 37, 343-351). In the absence of the T7 1.2 and E. coli optA gene products, the degradation of E. coli DNA proceeds normally, and T7 DNA synthesis is initiated at the primary origin. However, T7 DNA synthesis ceases prematurely and the newly synthesized DNA is degraded; no viable phage particles are released. The gene 1.2 protein has been purified to apparent homogeneity from cells in which the cloned 1.2 gene is overexpressed. Purification of the [35S] methionine-labeled protein was followed by monitoring the radioactivity of the protein and by gel electrophoresis. The purified protein has been identified as the product of gene 1.2 on the basis of molecular weight and partial amino acid sequence. We have found that extracts of E. coli optA1 cells infected with T7 gene 1.2 mutants are defective in packaging exogenous T7 DNA when such extracts are prepared late in infection. Purified gene 1.2 protein restores packaging activity to these defective extracts, thus providing a biological assay for gene 1.2 protein. No specific enzymatic activity has been found associated with the purified gene 1.2 protein.     

Bacteriophage lambda DNA packaging in vitro. The involvement of the lambda FI gene product, single-strand DNA, and a novel lambda-directed protein in the packaging reaction

The FI gene product (gp) of bacteriophage lambda is required during phage head assembly in vivo. Mutations in this gene lead to an accumulation of immature concatemeric lambda DNA and of proheads that appear normal and are competent for DNA packaging in vitro. This phenotype can be taken as evidence of a failure to couple DNA and proheads for packaging/maturation. In contrast to the requirement for gpFI in vivo, the packaging of lambda DNA in vitro occurs efficiently in the complete absence of gpFI. However, if ssDNA is included at the outset of the in vitro packaging reaction, DNA packaging is blocked. This block to packaging is relieved by addition of gpFI. Thus packaging of lambda DNA in vitro can be made dependent of gpFI by the inclusion of ssDNA at the outset of the reaction. Inhibition of DNA packaging by ssDNA appears to be mediated by a lambda b region-directed protein (packaging inhibitor, ben protein) that is present in the crude extracts of cells used to support the early steps of the packaging reaction. Neither ssDNA nor the packaging inhibitor alone has significant inhibitory effect on packaging; both components are required together to effect the inhibition that is relieved by gpFI. The packaging inhibitor was extensively purified and shown to have endonucleolytic activity. Several lines of evidence are presented to support the idea that both the inhibitory and endonucleolytic activities are functions of the same protein. Although gpFI relieves the inhibition imposed by the ben protein in packaging, gpFI fails to block the DNA cleavage activity of the ben protein in the standard endonuclease assay.     

The Nu1 subunit of bacteriophage lambda terminase

The maturation and packaging of bacteriophage lambda DNA are catalyzed by the phage terminase enzyme. Terminase is composed of two protein subunits, gpNu1 and gpA. The holoenzyme is multifunctional in vitro; it binds to and cleaves lambda DNA at the cos site (where cos represents cohesive-end site), packages DNA into lambda proheads, and is also a DNA-dependent ATPase. The genes of the two subunits have been cloned separately into powerful expression vectors which allow for very high levels of protein overproduction. The gpNu1 protein has been purified to homogeneity and has a monomeric molecular weight of 21,200, in close agreement with the Mr of 20,444 expected from its amino acid sequence. Both gel filtration and sedimentation velocity centrifugation indicate that the native gpNu1 protein exists as a Mr greater than 500,000 aggregate. The sequence of the first 20 amino acids and the overall composition both match those predicted by the nucleotide sequence of the Nu1 gene. Purified gpNu1 is able to complement gpA-containing extracts in both lambda DNA packaging and cos cleavage assays. The Nu1 gene amino acid sequence predicts DNA binding by the protein, and gpNu1 does show specific binding to lambda DNA by filter binding assays. Also, as predicted from its sequence, gpNu1 exhibits ATPase activity; but in contrast to the holoenzyme, this activity is DNA-independent.     

Rescue of abortive T7 gene 2 mutant phage infection by rifampin.

Infection of Escherichia coli with T7 gene 2 mutant phage was abortive; concatemeric phage DNA was synthesized but was not packaged into the phage head, resulting in an accumulation of DNA species shorter in size than the phage genome, concomitant with an accumulation of phage head-related structures. Appearance of concatemeric T7 DNA in gene 2 mutant phage infection during onset of T7 DNA replication indicates that the product of gene 2 was required for proper processing or packaging of concatemer DNA rather than for the synthesis of T7 progeny DNA or concatemer formation. This abortive infection by gene 2 mutant phage could be rescued by rifampin. If rifampin was added at the onset of T7 DNA replication, concatemeric DNA molecules were properly packaged into phage heads, as evidenced by the production of infectious progeny phage. Since the gene 2 product acts as a specific inhibitor of E. coli RNA polymerase by preventing the enzyme from binding T7 DNA, uninhibited E. coli RNA polymerase in gene 2 mutant phage-infected cells interacts with concatemeric T7 DNA and perturbs proper DNA processing unless another inhibitor of the enzyme (rifampin) was added. Therefore, the involvement of gene 2 protein in T7 DNA processing may be due to its single function as the specific inhibitor of the host E. coli RNA polymerase.     

Isolation of the gene encoding yeast DNA polymerase I

A yeast genomic DNA expression library in lambda gt11 antibody prepared against yeast DNA polymerase I were used to isolate the gene encoding DNA polymerase I. The identity of the DNA polymerase I gene was determined by several criteria. First, the clone-encoded protein is immunologically related to DNA polymerase I. Second, cells containing the gene cloned in the high copy number plasmid YEp24 overproduce the polymerase activity 4- to 5-fold as measured in yeast extracts. Finally, insertion of the gene downstream from a bacteriophage T7 promoter allows synthesis of yeast DNA polymerase I in Escherichia coli. Gene disruption and Southern hybridization experiments show that the polymerase is encoded by an essential, single copy gene. Examination of the germinated spores containing the disrupted gene reveals a defect in nuclear division and a terminal phenotype typical of replication mutants.     

Multidimensional analysis of intracellular bacteriophage T7 DNA: effects of amber mutations in genes 3 and 19.

By use of rate-zonal centrifugation, followed by either one- or two-dimensional agarose gel electrophoresis, the forms of intracellular bacteriophage T7 DNA produced by replication, recombination, and packaging have been analyzed. Previous studies had shown that at least some intracellular DNA with sedimentation coefficients between 32S (the S value of mature T7 DNA) and 100S is concatemeric, i.e., linear and longer than mature T7 DNA. The analysis presented here confirmed that most of this DNA is linear, but also revealed a significant amount of circular DNA. The data suggest that these circles are produced during DNA packaging. It is proposed that circles are produced after a capsid has bound two sequential genomes in a concatemer. The size distribution of the linear, concatemeric DNA had peaks at the positions of dimeric and trimeric concatemers. Restriction endonuclease analysis revealed that most of the mature T7 DNA subunits of concatemers were joined left end to right end. However, these data also suggest that a comparatively small amount of left-end to left-end joining occurs, possibly by blunt-end ligation. A replicating form of T7 DNA that had an S value greater than 100 (100S+ DNA) was also found to contain concatemers. However, some of the 100S+ DNA, probably the most branched component, remained associated with the origin after agarose gel electrophoresis. It has been found that T7 protein 19, known to be required for DNA packaging, was also required to prevent loss, probably by nucleolytic degradation, of the right end of all forms of intracellular T7 DNA. T7 gene 3 endonuclease, whose activity is required for both recombination of T7 DNA and degradation of host DNA, was required for the formation of the 32S to 100S molecules that behaved as concatemers during gel electrophoresis. In the absence of gene 3 endonuclease, the primary accumulation product was origin-associated 100S+ DNA with properties that suggest the accumulation of branches, primarily at the left end of mature DNA subunits within the 100S+ DNA.     

Gene 1.2 protein of bacteriophage T7. Effect on deoxyribonucleotide pools

The gene 1.2 protein of bacteriophage T7, a protein required for phage T7 growth on Escherichia coli optA1 strains, has been purified to apparent homogeneity and shown to restore DNA packaging activity of extracts prepared from E. coli optA1 cells infected with T7 gene 1.2 mutants (Myers, J. A., Beauchamp, B. B., White, J. H., and Richardson, C. C. (1987) J. Biol. Chem. 262, 5280-5287). After infection of E. coli optA1 by T7 gene 1.2 mutant phage, under conditions where phage DNA synthesis is blocked, the intracellular pools of dATP, dTTP, and dCTP increase 10-40-fold, similar to the increase observed in an infection with wild-type T7. However, the pool of dGTP remains unchanged in the mutant-infected cells as opposed to a 200-fold increase in the wild-type phage-infected cells. Uninfected E. coli optA+ strains contain severalfold higher levels of dGTP compared to E. coli optA1 cells. In agreement with this observation, dGTP can fully substitute for purified gene 1.2 protein in restoring DNA packaging activity to extracts prepared from E. coli optA1 cells infected with T7 gene 1.2 mutants. dGMP or polymers containing deoxyguanosine can also restore packaging activity while dGDP is considerably less effective. dATP, dTTP, dCTP, and ribonucleotides have no significant effect. The addition of dGTP or dGMP to packaging extracts restores DNA synthesis. Gene 1.2 protein elevates the level of dGTP in these packaging extracts and restores DNA synthesis, thus suggesting that depletion of a guanine deoxynucleotide pool in E. coli optA1 cells infected with T7 gene 1.2 mutants may account for the observed defects.     

Role of gene 6 exonuclease in the replication and packaging of bacteriophage T7 DNA

When bacteriophage T7 gene 6 exonuclease is genetically removed from T7-infected cells, degradation of intracellular T7 DNA is observed. By use of rate zonal centrifugation, followed by either pulsed-field agarose gel electrophoresis or restriction endonuclease analysis, in the present study, the following observations were made. (1) Most degradation of intracellular DNA requires the presence of T7 gene 3 endonuclease and is independent of DNA packaging; rapidly sedimenting, branched DNA accumulates when both the gene 3 and gene 6 products are absent. (2) A comparatively small amount of degradation requires packaging and occurs at both the joint between genomes in a concatemer and near the left end of intracellular DNA; DNA packaging is only partially blocked and end-to-end joining of genomes is not blocked in the absence of gene 6 exonuclease. (3) Fragments produced in the absence of gene 6 exonuclease are linear and do not further degrade; precursors of the fragments are non-linear. (4) Some, but not most, of the cleavages that produce these fragments occur selectively near two known origins of DNA replication. On the basis of these observations, the conclusion is drawn that most degradation that occurs in the absence of T7 gene 6 exonuclease is caused by cleavage at branches. The following hypothesis is presented: most, possibly all, of the extra branching induced by removal of gene 6 exonuclease is caused by strand displacement DNA synthesis at the site of RNA primers of DNA synthesis; the RNA primers, produced by multiple initiations of DNA replication, are removed by the RNase H activity of gene 6 exonuclease during a wild-type T7 infection. Observation of joining of genomes in the absence of gene 6 exonuclease and additional observations indicate that single-stranded terminal repeats required for concatamerization are produced by DNA replication. The observed selective shortening of the left end indicates that gene 6 exonuclease is required for formation of most, possibly all, mature left ends.     

Sites and gene products involved in lambdoid phage DNA packaging.

21 is a temperate lambdoid coliphage, and the genes that encode the head proteins of lambda and 21 are descended from a common ancestral bacteriophage. The sequencing of terminase genes 1 and 2 of 21 was completed, along with that of a segment at the right end of 21 DNA that includes the R4 sequence. The R4 sequence, a site that is likely involved in termination of DNA packaging, was found to be very similar to the R4 sequences of lambda and phi 80, suggesting that R4 is a recognition site that is not phage specific. DNA packaging by 21 is dependent on a host protein, integration host factor. A series of mutations in gene 1 (her mutations), which allow integration host factor-independent DNA packaging by 21, were found to be missense changes that affect predicted alpha-helixes in gp1. gp2, the large terminase subunit, is predicted to contain an ATP-binding domain and, perhaps, a second domain important for the cos-cutting activity of terminase. orf1, an open reading frame analogous in position to FI, a lambda gene involved in DNA packaging, shares some sequence identity with FI. orf1 was inactivated with nonsense and insertion mutations; these mutations were found not to affect phage growth. 21 was also not able to complement a lambda FI mutant.     

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.     

Bacteriophage T7 DNA packaging. I. Plasmids containing a T7 replication origin and the T7 concatemer junction are packaged into transducing particles during phage infection

Bacteriophage T7 DNA is a linear duplex molecule with a 160 base-pair direct repeat (terminal redundancy) at its ends. During replication, large DNA concatemers are formed, which are multimers of the T7 genome linked head to tail through recombination at the terminal redundancy. We define the sequence that results from this recombination, a mature right end joined to the left end of T7 DNA, as the concatemer junction. To study the processing and packaging of T7 concatemers into phage particles, we have cloned the T7 concatemer junction into a plasmid vector. This plasmid is efficiently (at least 15 particles/infected cell) packaged into transducing particles during a T7 infection. These transducing particles can be separated from T7 phage by sedimentation to equilibrium in CsCl. The packaged plasmid DNA is a linear concatemer of about 40 x 10(3) base-pairs with ends at the expected T7 DNA sequences. Thus, the T7 concatemer junction sequence on the plasmid is recognized for processing and packaging by the phage system. We have identified a T7 DNA replication origin near the right end of the T7 genome that is necessary for efficient plasmid packaging. The origin, which is associated with a T7 RNA polymerase promoter, causes amplification of the plasmid DNA during T7 infection. The amplified plasmid DNA sediments very rapidly and contains large concatemers, which are expected to be good substrates for the packaging reaction. When cloned in pBR322, a sequence containing only the mature right end of T7 DNA is sufficient for efficient packaging. Since this sequence does not contain DNA to the right of the site where a mature T7 right end is formed, it was expected that right ends would not form on this DNA. In fact, with this plasmid the right end does not form at the normal T7 sequence but is instead formed within the vector. Apparently, the T7 packaging system can also recognize a site in pBR322 DNA to produce an end for packaging. This site is not recognized solely by a "headful" mechanism, since there can be considerable variation in the amount of DNA packaged (32 x 10(3) to 42 x 10(3) base-pairs). Furthermore, deletion of this region from the vector DNA prevents packaging of the plasmid. The end that is formed in vector DNA is somewhat heterogeneous. About one-third of the ends are at a unique site (nucleotide 1712 of pBR322), which is followed by the sequence 5'-ATCTGT-3'. This sequence is also found adjacent to the cut made in a T7 DNA concatemer to produce a normal T7 right end.     

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.     

Deoxyribonucleic acid polymerase of bacteriophage T7. Purification and properties of the phage-encoded subunit, the gene 5 protein.

DNA polymerase of bacteriophage T7 is composed of two subunits, the gene 5 protein of the phage and the host-specified thioredoxin. The gene 5 protein has been purified 7400-fold to homogeneity from bacteriophage T7-infected Escherichia coli 7400 trxA cells that lack thioredoxin. The purification procedure has been monitored by using a complementation assay in which thioredoxin interacts with the gene 5 protein to form an active DNA polymerase. The purified gene 5 protein is a single polypeptide having a molecular weight of 87,000. The gene 5 protein itself has only 1 to 2% of the polymerase activity of T7 DNA polymerase. However, T7 DNA polymerase can be reconstituted by the addition of homogeneous thioredoxin to the gene 5 protein. Optimal reconstitution is obtained when the molar ratio of thioredoxin/gene 5 protein is 150. Under these conditions, the gene 5 protein attains approximately 80% of the activity of an equal amount of T7 DNA polymerase. The apparent Km for thioredoxin in the reaction to restore DNA polymerase activity is 2.8 x 10(-8) M. The enzymatic properties of the reconstituted enzyme are indistinguishable from those of T7 DNA polymerase synthesized in vivo; the reconstituted polymerase interacts with T7 gene 4 protein to catalyze DNA synthesis on duplex DNA templates.