From the Department of Biochemistry and Molecular Biology, University of Maryland Medical School, Baltimore, Maryland 21201-1503
Received for publication, August 21, 2002, and in revised form, November 25, 2002
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ABSTRACT |
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Phage T4 terminase is a two-subunit enzyme that
binds to the prohead portal protein and cuts and packages a
headful of concatameric DNA. To characterize the T4 terminase
large subunit, gp17 (70 kDa), gene 17 was cloned and expressed as a
chitin-binding fusion protein. Following cleavage and release of gp17
from chitin, two additional column steps completed purification. The
purification yielded (i) homogeneous soluble gp17 highly active in
in vitro DNA packaging (~10% efficiency,
>108 phage/ml of extract); (ii) gp17 lacking endonuclease
and contaminating protease activities; and (iii) a DNA-independent
ATPase activity stimulated >100-fold by the terminase small subunit,
gp16 (18 kDa), and modestly by portal gp20 and single-stranded binding protein gp32 multimers. Analyses revealed a preparation of highly active and slightly active gp17 forms, and the latter could be removed
by immunoprecipitation using antiserum raised against a denatured form
of the gp17 protein, leaving a terminase with the increased specific
activity (~400 ATPs/gp17 monomer/min) required for DNA
packaging. Analysis of gp17 complexes separated from gp16 on glycerol
gradients showed that a prolonged enhanced ATPase activity persisted
after exposure to gp16, suggesting that constant interaction of the two
proteins may not be required during packaging.
Many dsDNA1
bacteriophage require the activity of two "terminase" proteins to
cut and package concatameric DNA into assembled phage proheads. In T4,
the gp16 (small subunit) and gp17 (large subunit) terminase proteins
are essential proteins for DNA packaging. (Similar proteins are gpA and
gpNu1 in The T4 terminase large subunit protein, gp17, has proven difficult to
purify because of the protein's limited solubility and toxicity to
host cells (14). Coexpression with the gp16 small subunit reduces
toxicity; thus, gp17 could be purified with gp16 in low amounts from a
temperature-induced expression vector (4). A vector that makes gp17
with a 20-amino acid OmpT leader peptide results in low amounts of
uninduced, soluble, and active protein, whereas the overexpressed
protein is insoluble and inactive and resists renaturation (15). A
His-tagged gp17 protein was also overexpressed and showed similar
problems in protein solubility as well as amino acid changes, possibly
to reduce toxicity.2 However,
Leffers and Rao (5) overcame these problems by modifying the growth
conditions and strictly controlling the basal level expression from a
His-tagged cloned gene.
Although gp17 terminase is specifically assayed by its ability to make
phage by in vitro DNA packaging and to bind and hydrolyze ATP (4, 5, 16), it was unclear whether its ATPase and nuclease
activities require interaction with additional nucleic acid or protein
factors. To address these issues and to highly purify gp17, we employed
three column steps, including the use of dsDNA- and ssDNA-cellulose to
separate gp17 from other nucleic acid-interacting proteins.
The purified gp17 terminase ATPase activity is stimulated by
interactions with phage T4 proteins gp32 and gp20 and, most strongly, with the terminase small subunit, gp16. We also discovered that the
enzyme preparation was composed of at least two forms of the protein, a
form that could be activated by gp16 to a high turnover ATPase and a
less active form separable by immunoprecipitation with antiserum
prepared against denatured gp17. After removal of low activity protein,
ATPase activity of 400 ATPs/gp17/min remained, the highest activity
reported for any large subunit terminase. These experiments suggest
that significant structural differences allow separation of the two
forms and also show that terminase undergoes a major conformational
change to form a gp16-induced multimer whose high activity is not
dependent upon continued interaction with gp16. The findings advance
our understanding of terminase interactions and conformational changes
that lead to the catalytically active packasome.
Chemicals were reagent-grade, and enzymes were used as
recommended by the suppliers. Routine cloning was performed as
described (24). DNA concentrations were estimated using a
spectrophotometric determination at 260 and 280 nm as described (25).
Protein determinations were made by the Bradford procedure using
commercially available reagents (Bio-Rad).
Gene 17 Cloning and Expression: Construction of Vector
pT5--
Full-length active gp17 was purified based on the Impact
system of cloning (New England Biolabs, Inc.). To clone the wild-type gene 17 sequence into vector pCYB2 (6843 bp), a cloning method was
devised employing PCR primers 1 (5'-GACGGGTAACCCTGTCGACTCATATGGAACAACCGATTAATGT-3') and 2 (3'-GTACCATACAGTTACCATGGGCCCCCT-5'). Primer 1 was designed for the 5'-end of the gene and introduced an
NdeI site immediately at the start of gene 17 (boldface).
Primer 2 was designed for the 3'-end of the gene and replaced the stop
codon of gene 17 with a SmaI site (boldface). Purified
wild-type T4 DNA served as template for PCR, and a 1851-bp fragment was
isolated, digested with NdeI/SmaI, and purified.
This fragment was ligated into NdeI/SmaI-cut pCYB2, yielding vector pH5 (8634 bp). This original tac
promoter-containing vector was electroporated into
Escherichia coli DH5 gp17 Purification--
Large-scale cultures were done at the
University of Maryland Bioprocess Facility (College Park, MD). 100 liters of HMS174(DE3)/pT5 were grown to A600 nm = 0.6, induced for 3 h with 0.5 mM isopropyl- SDS-PAGE/Blotting--
SDS-PAGE followed by Coomassie
Brilliant Blue R-250 protein staining or by blotting using a
horseradish peroxidase-conjugated secondary antibody (Bio-Rad) for
fluorescence detection of primary antibody bound to protein was done
using standard Western blot protocols (25).
N-terminal Sequencing of gp17--
Automated Edman
sequencing of the purified full-length 70-kDa gp17 protein and the
sample fragmented to 65 kDa was done over six cycles directly
from solution samples at the Johns Hopkins University Synthesis and
Sequencing Facility.
In Vitro DNA Packaging--
In vitropackaging was
performed as previously described (17).
Radioactive ATPase Assays--
Purified gp17 and gp16 proteins
were preincubated with an equal volume of 8% polyethylene glycol
before addition of the same volume of 2× ATPase buffer to start the
reaction. The final reaction buffer was 100 mM Tris-HCl, pH
8.0, 10 mM MgCl2, 10 mM DTT, 0.1 M NaCl, 1 mM spermidine HCl, 0.1 mM
putrescine, 1 mM ATP, and 10 µCi of
[ Nuclease Assays--
DNA cleavage assays were carried out in
numerous ways, typically using a reaction mixture that finally
contained 20 mM Tris-HCl, pH 8.0, 5 mM
MgCl2, 80 mM KCl, 2 mM ATP, 2 mM spermidine, and 2.7% polyethylene glycol (PEG).
Mixtures of the terminase proteins (1.5 µg of gp17 + 0.5-1.5 µg of
gp16) in 15 µl of buffer were made, and the reactions were started by
addition of 4 µg of DNA. Various DNA substrates were analyzed,
including linear marker DNAs and plasmid DNAs such as pCBR2 and pR67
(4), which contain the gene 16-17 sequence, and pDH72DE3 (29), a kind
gift of Dr. E. P. Geiduschek. Reactions were incubated at 30 °C
for 3 h and heated at 65 °C for 10 min prior to 0.8% agarose
gel electrophoresis and ethidium bromide staining following standard
methods (25).
Native Agarose Gel Electrophoresis of Proteins--
As in the
study of large viral protein assemblies (22), samples were run in TAMg
buffer (1× = 40 mM Tris base, 20 mM acetic acid, pH 8.1, and 1 mM MgSO4) with 10 mM MgSO4. Gels were run at 70-90 V (20-40 mA)
from 19 to 22 °C, and the pH of the buffer was kept constant.
Ferguson plot and log-log graph analyses of the gel data were done as
described (23), assuming negligible effects of concaving Ferguson plot
data due to the changes in agarose concentration from 0.75 to
2.25%.
Glycerol Gradient Separation of Proteins--
Glycerol gradients
were made inside 5 × 41-mm polyethylene tubes by placing 300 µl
of 40% glycerol in Q buffer underneath 300 µl of 10% glycerol in Q
buffer and forming the gradient using a Biocomp Gradient Master 105 gradient maker set at 86° and 46 s, run at 22 rpm, and
equilibrated to 4 °C. Up to 60 µl of sample could be loaded and
run at 30,000 × g in an SW 50.1 swinging bucket rotor
overnight at 4 °C for 15-20 h. Fractions were collected using a
modified 25-gauge (5/8) needle pierced through the bottom of the
tube to collect drops.
Immunoprecipitation of Proteins--
Immunoprecipitation was
carried out as described by Springer (27). Protein A-Sepharose
(Amersham Biosciences) was washed as a 50% slurry in dilution buffer
(10 mM Tris-HCl, pH 8.0, 140 mM NaCl, 0.1%
Triton X-100, 0.1% bovine serum albumin, and 0.025% sodium azide) and
kept at 4 °C prior to use. For coupling, proteins were diluted in
200 µl of buffer; antibody from serum (usually 1-5 µl) was added;
and the mixture was rocked at 4 °C for 1.5 h. 10-40 µl of
50% protein A-Sepharose slurry were added, and the mixture was rocked
for another 1.5 h at 4 °C. The mixture was centrifuged at low
speed at 200× g, and the supernatant was carefully removed.
The pellet was washed repeatedly with dilution buffer and, for gel or
titer analysis, brought up in a minimum volume (50 or 100 µl) of
buffer containing 5.8 g/liter NaCl, 2 g/liter
MgSO4·7H2O, 50 ml/liter 1 M
Tris-Cl, pH 7.5, and 5 ml/liter 2% gelatin; and a fraction of the
pellet slurry was analyzed.
Antisera and Purification of Anti-gp17 Antibody-containing
IgG--
The development of polyclonal antisera raised against native
and active gp16 (3) and against the denatured form of OmpT leader/gp17
terminase has been described previously by Lin (15), following a
protocol of eluting the SDS-PAGE-isolated proteins from gels (28). To
purify the gp17 IgG from the other serum proteins, 1 ml of anti-gp17
antiserum was diluted to 5 ml in Q buffer and loaded onto a 1-ml
protein A column previously washed with 10 ml of water and 10 ml of 0.1 M sodium citrate, pH 3-4, and equilibrated in Q buffer.
After loading (0.5 ml/min), the column was washed with 10 ml of Q
buffer. A large non-binding fraction of serum proteins was saved for
further analysis. For IgG elution, 0.1 M sodium citrate, pH
3.5, was added to the column, and IgG eluting in a single sharp peak
was immediately neutralized to pH 7.0 with 2 M NaOH
(~30-35 µl/1-1.5 ml of sodium citrate/antibody wash). This
antibody fraction was not stimulatory for the gp17 ATPase until further
dialyzed from sodium citrate against Q buffer (with no ATP) and was
concentrated to its original volume of ~1 ml using a Centriprep C30
membrane concentrator. The serum protein fraction was also concentrated
to approximately the original serum concentration.
Treatment of the gp16-gp17 Terminase Complex with
Anti-gp17 Antiserum for Immunoprecipitation before Glycerol Gradient
Analysis--
Most terminase analyses started with a 1:1 or 1:1.5
(w/w) gp17/gp16 mixture (giving a 4-6-fold molar excess of gp16),
except where indicated. 30 µl (15 µg) of gp16 were first mixed with
15 µl of 12.5% PEG solution, and 15 µl (10 µg) of gp17 were
added and mixed by light pipetting and then mixing for 10 min at
4 °C. 7.5 µl of crude anti-gp17 antiserum were added, and the
tubes were rocked for 1 h. 50 µl of 50% protein A-Sepharose
slurry were added and incubated for 1 h with mixing; the tubes
were spun at 1500 rpm (200 × g); and the supernatant
was carefully removed for analysis. Generally, a small fraction of the
immunoprecipitation supernatant was loaded onto the glycerol
gradient, and 45-50-µl fractions were collected from the gradient.
Treatment of the gp16-gp17 Terminase Complex with Pure
Anti-gp17 Antibody-containing IgG before Glycerol Gradient
Analysis--
6.6 µg (10 µl) of purified gp17 protein were
incubated with 5 µg (10 µl) of gp16 protein for 30 min; 30 µl of
purified anti-gp17 antibody were added; and the reaction was rotated
for 1 h. The solution was placed on top of a 600-µl glycerol
gradient (10-40%) and separated by spinning at 40,000 rpm for 5 h.
Densitometry--
SDS-PAGE or Western blot data were analyzed
using the AlphaImager 2000 documentation and analysis system. Most data
were visualized using the trans-white illumination apparatus
and quantitated using AlphaImager Version 3.3b software (Alpha Innotech
Corp.).
Purification of Active Soluble gp17: Expression of a
gp17-Intein/CBD Fusion Protein--
Purification of the
gp17 terminase large subunit was based on the New England Biolabs
intein/CBD fusion protein method. Gene 17 was cloned into the pTYB2
fusion vector to make vector pT5, which expresses the gp17-intein/CBD
fusion. In the cell lysate, this fusion protein was >75% soluble and
produced in large amounts as detected by SDS-PAGE and Western blotting
using anti-gp17 antibody (Fig.
1A). Significant accumulation
of fusion protein was detected after 3 h of induction with
isopropyl- Slow Proteolysis of gp17 in Chitin Column Fractions--
gp17 from
the chitin column is apparently homogeneous as determined by SDS-PAGE
(Fig. 1B). Nevertheless, we discovered that gp17 in
fractions eluted from the chitin column was slowly converted to 68- and
65-kDa forms if stored at 4 °C without further purification. In Fig.
2, sample A represents the
full-length chitin-purified protein, which was slowly converted to
sample B. The samples were N-terminally sequenced, and the
results of this analysis (Fig. 2, boldface) confirmed that
sample A was the full-length gp17 (70 kDa) and that the
transition to sample B (65 kDa) resulted from cleavage of
gp17 at two distinct positions in its C terminus, both of which were
identified within the gp17 sequence by N-terminal sequencing of six
amino acid residues of the released peptides.
Further Chromatographic Purification of gp17--
We also found
that gp17 did not bind to ssDNA-cellulose (4) or dsDNA-cellulose (this
work), and the use of these media removed not only the proteolytic
activity, but also contaminating nucleases present in the preparation
that interfered with subsequent analysis. In fact, washing
ssDNA/dsDNA-cellulose after gp17 elution with 1 M NaCl and
1% Triton X-100 and concentrating showed that E. coli
proteins, some close in size to gp17 and some that cross-reacted with
anti-gp17 antibody upon longer exposure of Western blots, were purified
from the gp17 sample (data not shown). As a final step, gp17 was bound
to blue Sepharose and washed with 1 M NaCl or 50 mM ATP and was not removed from the column until elution at
both concentrations of NaCl and ATP (4). After elution, the protein was
dialyzed thoroughly in storage buffer and stored unfrozen at Analysis of Purified gp17: In Vitro Packaging of T4 DNA with
Purified gp17 and gp16 Proteins--
The packaging of T4 DNA in
vitro is the definitive test of the identity and functionality of
the terminase proteins (4, 16, 17). In this assay, extracts are made
with 16am17am Nuclease Activity Assay--
Numerous attempts were made to
determine whether gp17 alone or with T4 proteins with which it is known
to interact can cleave DNA in vitro. Various plasmids and
other linear DNAs have been analyzed, including some with the gene 16 sequence identified as the location of a Pac site (3); and
thus far, none have been cleaved significantly by gp17 or by gp17
co-incubated with other T4 proteins (gp20, gp16, and gp32). In numerous
experiments, no significant nicking or cutting of various plasmid DNAs
was seen for the terminase proteins compared with control buffer with or without co-incubation with the other T4 proteins, even when gp17 and
gp16 were added at >10-fold molar excess compared with plasmid DNA.
ATPase of the Purified gp17 Terminase Large Subunit: Qualitative
Effects--
Purified gp17 was found to contain an intrinsic ATPase
activity (1 ATP hydrolyzed/gp17/min) that could be significantly
stimulated (~100-fold) by addition of the gp16 small subunit
terminase protein (120 ATPs hydrolyzed/gp17/min). The gp16 protein
itself was previously shown to lack ATPase activity (3). Subjecting
gp17 to numerous freeze/thaw cycles did not decrease the ATPase
activity of the preparation, although gp17 showed a half-life at
37 °C of ~1 h. Addition of the polymer PEG (~20 kDa) at 8%
(w/v) to the terminase proteins increased the activity seen, presumably
by promoting protein interactions. The purified terminase subunit
lacked detectable nucleic acid, and addition of numerous nucleic acids
failed to stimulate the ATPase. These included wild-type T4 DNA cut
with restriction enzymes or uncut;
We also tested the effect of gp20 portal addition on the ATPase
hydrolysis rate of gp17 plus gp16. A number of different portal protein-containing preparations were analyzed, including purified gp20
dodecamer (18), gp20-green fluorescent protein fusion dodecamer (19),
an alternatively purified gp20 protein (20), proheads, and a 24-mer
peptide of gp20 identified to interact with gp17 (21). When testing
gp20 or gp20-green fluorescent protein fusion dodecamers, consistently
higher (up to ~20%) ATPase hydrolysis rates were achieved (data not
shown). Proheads or the monomeric gp20 preparation (20) consistently
showed no stimulatory effects. Addition of the gp20 peptide inhibited
the ATPase slightly, but only at great molar excess. Because
interaction of gp17 with the T4 single-stranded binding protein gp32
has been demonstrated (20), we investigated the effects of gp32 on the
gp17 ATPase in the presence of various nucleic acid substrates. Under
conditions of limiting amounts of gp17 and saturating amounts of gp16,
addition of gp32 with ssDNA consistently increased (approximately
doubled) the ATPase hydrolysis rate of the gp16-gp17 terminase complex. Addition of RNA with gp32 also increased the rate of ATP hydrolysis, whereas T4 wild-type DNA addition with gp32 had no effect on the hydrolysis rate.
Stimulation of gp17 ATPase with gp16 and Affinity of Terminase for
ATP--
To examine the effect of gp16 addition on the gp17 ATPase,
ATPase reactions were assayed with fixed amounts of gp17 and ATP with
varying amounts of gp16 added. Addition of gp16 to gp17 at submolar to
equimolar amounts dramatically increased the initial ATPase hydrolysis
rates observed (Fig. 3A).
Above equimolar amounts, the initial ATPase hydrolysis rates increased,
but further increases in the amounts of gp16 added in great molar
excess (from 10- to 60-fold) had only minor effects on the ATPase
hydrolysis rate of gp17 (Fig. 3B), suggesting that the
stimulation was saturable. From this analysis, we were able to
determine the maximum effect on initial reaction velocity that gp16
would have on gp17 under these reaction conditions and derive a
double-reciprocal plot showing a linear correlation for the rate of
reaction versus the concentration of gp16 protein (data not
shown). From this plot, the KD for gp16 binding to
0.7 µM gp17 was determined to be 5 µM
(relatively weak binding of the two terminase subunits), consistent
with previous measurements as well as additional work presented below
(4, 20).
To determine the affinity with which the gp17 ATPase binds ATP, we
determined the initial rates of ATP hydrolysis at varying concentrations of ATP with fixed concentrations of the terminase large
subunit. Two concentrations of the gp16 small subunit were examined, 7- and 21-fold molar excesses of gp16, the latter near saturation. As Fig.
3C shows, increasing the concentration of ATP increased the
initial rates of hydrolysis. Incubation of the gp17 ATPase with
increasing amounts of gp16 had a positive effect on the initial rates
of ATP hydrolysis (5.6 versus 16.7 µM gp16). Both data sets showed increased initial rates, which plateaued beyond
addition of 5 mM ATP; and the maximal velocities achieved differed according to the amount of gp16 added to the reaction. The
highest initial velocities were achieved at saturating concentrations of gp16 and surpassed 130 ATPs hydrolyzed/gp17/min. Lineweaver-Burk plot (double-reciprocal) analysis of these data showed that the primary
effect of the gp16 addition to the ATPase was to increase the
Vmax of the reaction and that the affinity of
the enzyme for ATP was not changed due to the increased presence of
gp16, as reflected by the same Km (380 µM).
Effect of Anti-gp17 Antiserum on the gp16-gp17 Complex
ATPase--
In an effort to inhibit the gp17 ATPase with anti-gp17
antibody, anti-gp17 antiserum was mixed with the gp17 protein and
incubated for 1 h on ice either before or after addition of gp16.
To our surprise, in both cases, addition of anti-gp17 antiserum
significantly increased the gp17 ATPase hydrolysis rate (Fig.
4A). Anti-gp17 antiserum had
no detectable ATPase activity itself, and preimmune serum or
nonspecific serum did not show such stimulation. IgG purified from
anti-gp17 antiserum also stimulated the ATPase hydrolysis rate. In
contrast, the anti-gp17 antiserum proteins depleted of IgG showed no
stimulation of the gp17 ATPase or any detectable ATPase activities when
assayed alone (Fig. 4B). The enhanced gp17 ATPase activity
assayed in the presence of antiserum was dependent upon addition of
gp16. To examine the effect of antibody addition on the gp17 ATPase
activity, a kinetic analysis was performed similar to the experiments
used to determine the Km for ATP. The initial rates
of ATP hydrolysis for gp17 were determined, with and without anti-gp17
antiserum, for two different subsaturating concentrations of gp16 while
varying the concentration of ATP. The concentration of gp17 used for
the experiments was kept constant at 0.8 µM. Addition of
antibody to both a 2.5-fold excess of gp16 (1.9 µM) (Fig.
4C) and a 7-fold excess of gp16 (5.5 µM) (data not shown) resulted in additional increases in the initial ATP hydrolysis rates seen for the increasing concentrations of ATP. Interestingly, the maximal rate stimulation seen with the two gp16
concentrations was achieved at about the same level (~190 ATPs
hydrolyzed/gp17/min) in both experiments, suggesting that antibody
addition raised the effective level of gp16 to saturation (comparable
Vmax levels for antibody addition in both
cases). The Lineweaver-Burk plot analysis of these data (not shown)
showed that, in both cases, the affinity of gp17 for ATP was not
changed upon antibody addition and that the main effect of antibody
addition (similar to gp16 addition) was to increase the
Vmax of the reaction, with no effect on the
Km for ATP.
Immunoprecipitation Experiments with Anti-gp17 Antiserum--
We
supposed either that the enhancement of gp17 ATPase might result from
the direct interaction of antibody with functional gp17 protein,
somehow stimulating its activity, or that the antibody might interact
primarily with nonfunctional forms of the gp17 protein to prevent their
inhibitory interactions with the fraction of active gp17 present in the
preparation. Immunoprecipitation experiments on gp17 or gp16-gp17
favored the latter possibility because the bulk of the ATPase activity
could not be immunoprecipitated by addition of increasing amounts of
anti-gp17 antiserum and protein A-Sepharose. gp17 and gp16-gp17
immunoprecipitation experiments showed that nearly all of the gp17
ATPase activity remained in the supernatant after immunoprecipitation
and that the ATPase showed continued dependence on addition of gp16,
whether or not anti-gp17 antiserum was added. An analogous experiment
was performed using anti-gp16 antibody to immunoprecipitate the gp16
protein alone or just after addition to gp17 on ice prior to performing the ATPase assay. The results showed that the stimulatory activity of
gp16 was removed in both cases and that little or no gp17
ATPase-stimulating activity remained in the supernatant after
immunoprecipitation (data not shown). This supports previous
experimental evidence suggesting that interactions between the
two terminase proteins are essential for high gp17 ATPase activity
(30). Together, the data favor the hypothesis that anti-gp17 antibody
binds primarily the inactive gp17 protein and prevents its inhibitory
association with the active fraction of gp17 present.
To investigate this hypothesis, the distribution of both the ATPase
activity and gp17 protein in the supernatant and pellet was quantified.
The activity remaining in the supernatant could be increased with
increasing amounts of antibody, whereas the overall level of gp17
protein left in the supernatant was reduced, effectively increasing the
specific activity of the remaining gp17 protein. In this experiment
(Fig. 5, A-D), we determined the minimum amount of antibody that could be used to show stimulation of the gp17 ATPase. Addition of antiserum above 0.2 µl/µg of gp17 was sufficient to show a significant increase in the gp17 ATPase present in the supernatant after immunoprecipitation. As shown in Fig.
5 (compare A and B), only negligible ATPase
activity was found in the pellet after immunoprecipitation. However,
analysis of the gp17 protein in the supernatant and pellet fractions
showed that addition of increasing amounts of antiserum increased the pellet gp17, reduced the supernatant gp17, and also increased the
supernatant ATPase activity. Densitometric quantitation of the gp17
bands seen in the pellets and supernatants in Fig. 5C was
performed, and the averages of these results are given below the blot
(Fig. 5D). In this experiment, the most active fraction (Fig. 5C, SUPERNATANTS, lane 3)
contained only 10% of the original total gp17, thus yielding an ATPase
specific activity of 390 mol of ATP/gp17/min. The same results were
obtained using anti-gp17 antiserum or IgG purified from this serum. In
numerous experiments, increasingly active supernatant fractions had
lower levels of gp17 protein.
Analysis of Terminase Protein Interactions: Native TAMg Gel
Electrophoresis of gp17--
In an effort to demonstrate directly the
association of gp16 and gp17, native gel electrophoresis was performed
at low ionic strength (22). No interaction of the two proteins could be
demonstrated. However, the migration of the gp17 protein on the native
gels showed that, under low ionic strength conditions, the purified protein migrated as a single, relatively sharp band, consistent with a
high molecular mass complex of ~700 kDa determined by Ferguson plot
analysis (23). Different commercially available standards and
multimeric packasome proteins (gp16 rings and gp20 dodecamer) purified
and characterized in the laboratory yielded the expected molecular
masses in these plots (data not shown). Although it is intriguing that
the portal dodecamer (12 × 61 kDa) and the terminase large
subunit multimer showed comparable molecular masses in this procedure
(~700 kDa), the ATPase activity of gp17 electrophoresed or dialyzed
under the TAMg gel running buffer conditions showed reduced ATPase,
making it uncertain whether this complex actually represents the highly
active multimeric conformation of the protein.
Developing a Mini-glycerol Gradient Protocol for Small Volume
Analysis--
As an alternative and less harsh technique for probing
the interaction of gp16 and gp17 and to measure the molecular masses of
the active complexes, we developed a mini-glycerol gradient protocol
for analyzing samples in small volumes. Sedimentation of purified gp17
on these gradients under higher ionic strength (Q buffer) conditions
showed that the protein migrated as an apparent monomer as determined
in comparison with protein molecular mass standards (data not shown),
in contrast to our native TAMg gel results.
Analysis of gp16-gp17 and the gp16-gp17 Immunoprecipitation
Supernatant on Glycerol Gradients--
Incubation of gp17 with gp16
resulted in diffuse sedimentation of the gp17 protein throughout the
glycerol gradients, spanning molecular masses of 70-700 kDa or higher,
with the bulk of the protein loaded still resolving at the 70-kDa gp17
monomer position (Fig. 6A).
Although the gp16 and gp17 proteins did not co-sediment on the glycerol
gradient, the change in the sedimentation profile of gp17 suggests that
preincubation of the gp17 protein with gp16 had a significant effect on
the gp17 protein's ability to self-associate and to form multimers
that sedimented throughout the gradient; although as shown
in Fig. 6C, active forms of gp17 apparently still interacted
with inactive forms present in the preparation to disperse the active
forms. After immunoprecipitation of the gp16-gp17 complex with
anti-gp17 antiserum, which removed a significant fraction of low
activity and low molecular mass gp17, the supernatant showed that the
remaining high activity gp17 migrated as a well defined high molecular
mass complex in the lowest fractions of the glycerol gradient (Fig.
6B). Analysis of the high molecular mass gp17 protein
complex showed a significantly elevated ATPase activity (Fig.
6C, compare the fraction activity profiles in A and B). These results agreed with our earlier analyses and
suggested that removal of the antibody-binding gp17 fraction from the
preparation favored the formation of a high molecular mass complex
(700-900 kDa) with high activity.
Effects of Anti-gp17 Antibody Binding on High Molecular Mass gp17
Complex Formation--
To test the hypothesis that anti-gp17
antibody-containing IgG binds primarily inactive gp17, we incubated
gp17-gp16 or gp17 alone with anti-gp17 antibody-containing IgG prior to
performing the glycerol gradient separation of the mixture without
immunoprecipitation (Fig. 7, A
and B). These results showed that antibody addition resulted
in the diffuse sedimentation of gp17 throughout the fractions of the
gradient. However, Western blotting and ATPase analysis of the
fractions showed that an increasingly active gp17 protein sedimented
diffusely through the lower fractions of the glycerol gradient (Fig.
7C), presumably due to the binding (and sequestering from
interactions) of primarily inactive gp17 protein. In contrast to the
"terminase-alone" gradients (Fig. 6A), these fractions of gp17 sedimented in the presence of antibody showed that the diffusely sedimenting gp17 protein was active, with increasing ATPase
specific activities being found in fractions close to the bottom of the
gradient. With the bulk of the added IgG sedimenting at the top of the
gradients, where the activity was lowest, these results support the
notion that two forms of the gp17 protein exist in solution and that
the antibody preferentially binds non-active forms of the gp17 protein.
Consistent with what was found upon analysis of supernatants in
immunoprecipitation experiments, association of active forms of the
gp17 protein was favored to yield multimeric complexes with high
activity that sedimented toward the bottom of the
gradients. The data also suggest that the conversion between the two
forms present is a slow one because addition of antibody distinguishes
between the two forms that are separated by sedimentation. Interestingly, the ATPase analysis supported this "slow conversion" idea by showing that gp17 preincubated with gp16 had increasing gp16-independent ATPase activity for the fractions at the bottom of the
gradient. gp17 incubated with antibody alone showed no such
gp16-independent activity (Fig. 7, compare A and
B as assayed in C). We infer that preincubation
with gp16 resulted in a conformational change in the gp17 protein,
activating it into an ATP-hydrolyzing form that persisted for a long
time (for hours at 30 °C) even in the absence of gp16.
The intein/chitin fusion protein method of preparing phage T4
terminase overexpresses and purifies the gp17 subunit in soluble and
active form in essentially a single column step. Nevertheless, we found
that adequate terminase purification required two steps beyond
intein/chitin affinity chromatography. Without the use of
ssDNA/dsDNA-cellulose and blue Sepharose chromatography, strong DNA-binding E. coli proteins and proteases led to confusing
results in some assays. Thus, the chitin column-purified protein, even in the presence of protease inhibitors, underwent proteolysis, retaining partial ATPase activity, but releasing 12- and 39-amino acid
fragments from the C terminus. This proteolysis is analogous to how
certain preparations of the Our preparations of gp17 failed to bind ssDNA- or dsDNA-cellulose or to
hydrolyze numerous plasmid or phage DNAs tested at great molar enzyme
excess, readily allowing detection of a single DNA nick or cut per
enzyme molecule. Because the terminase large subunits display
the specific packaging endonuclease (7), and there is good evidence
that the T4 terminase large subunit hydrolyzes transcriptionally active
gene 17-containing plasmids in vivo (6, 14), other factors
appear to be necessary to achieve DNA-binding and DNA-directed
terminase activities in our preparation of gp17. We recently reported
that the gp17 terminase large subunit binds to the T4 late Although gp17 was found to have very low intrinsic ATPase activity (1 ATP hydrolyzed/gp17/min), several proteins stimulated this activity,
the gp16 terminase small subunit most dramatically. Addition of gp32
(single-stranded DNA-binding protein), which binds gp17 (20), doubled
the ATP hydrolysis rate, but only in the presence of ssDNA or RNA. The
portal protein gp20 also induced modest increases in the ATPase
hydrolysis rate. Interestingly, stimulation of gp17 ATPase apparently
requires that these proteins be multimers because monomeric gp20 and
gp32 had no effect. One explanation is that only interaction with the
multimers promotes high activity gp17 multimer formation.
The purified gp17 ATPase activity could be stimulated >100-fold by
addition of the gp16 terminase small subunit (130 ATPs hydrolyzed/gp17/min). Analysis showed that the affinity of the enzyme
for ATP was not changed with increasing gp16 concentrations; rather,
the primary effect on the enzyme was to increase the rate of catalysis
(Kcat). The Km for ATP was
calculated to be 380 µM. These values are in good
agreement with those (Km = 256 µM and
107 ATPs/gp17/min) reported by Leffers and Rao (5) and with ATPase
hydrolysis rates in other bacteriophage systems such as Anti-gp17 antiserum additionally increased the ATPase hydrolysis rate
of the gp16-dependent gp17 protein by at least 4-fold. Immunoprecipitation experiments demonstrated that both the total activity and the specific activity of the gp17 ATPase remaining in the
supernatant fractions were increased. Calculation of the ATPase
activity of gp17 remaining in the supernatant fraction in these
experiments showed that gp17 had a catalysis rate of ~400 ATPs
hydrolyzed/min/gp17, the highest activity reported for any
bacteriophage terminase and one that nears the activity it should
possess to account for packaging of DNA. Assuming that DNA packaging
rates in T3 and In an effort to determine whether the purified gp17 enzyme has a
tendency to form defined multimeric complexes, as has been shown for
two other identified packasome components (gp16 and gp20), native
agarose gel electrophoresis was performed on the sample under various
buffer conditions. Interestingly, running conditions were found in
which the protein did form a defined high molecular mass complex
estimated to be ~700 kDa in size. But because the protein had reduced
ATPase activity under the buffer conditions used, it was difficult to
assess the significance of this complex. A preliminary analysis of our
preparation by scanning transmission electron microscopy showed that
the preparation was composed of various molecular mass species from a
monomer-sized 70 kDa to >1
MDa.3 Consistent with these
observations, glycerol gradient analysis of gp17 showed that the
protein migrated at various multimeric molecular mass positions.
However, the highly active gp17 protein present in the supernatant
after immunoprecipitation resolved as a large molecular mass complex
toward the bottom of the gradient, with a molecular mass of 700-900
kDa in comparison with standards (Fig. 6B). Analysis showed
that antibody addition alone resulted in the sedimentation of higher
molecular mass gp17 complexes throughout the gradient, presumably by
removing the low activity forms of the protein from interaction.
Addition of gp16 to gp17 consistently resulted in more of the gp17
protein resolving as a higher activity and higher molecular mass
complex, suggesting that incubation with gp16 contributed to the
conversion to a specific, highly active multimeric form. In contrast,
it was observed that anti-gp16 antiserum abolished this stimulatory
effect of gp16 on gp17 ATPase activity. Interestingly, ATPase analysis
also showed that gp17 that was preincubated with gp16 had increasing
gp16-independent ATPase activity toward the bottom of the gradient,
where no gp16 was seen even on the longest exposure Western blots,
whereas gp17 that was not preincubated with gp16 did not (Fig.
7C). This is the first demonstration that the gp17 protein
can have enhanced ATPase activity in the absence of gp16. We infer that
preincubation with gp16 resulted in a conformational change in the gp17
protein, activating it into an ATP-hydrolyzing form that persisted for a long time (for hours at 30 °C) even in the absence of gp16. Slow
conversion between the high and low activity gp17 proteins is
consistent with their separation by immunoprecipitation. The possibility that the highly active gp17 ATPase depends upon continued interaction with gp17 IgG (e.g. to cross-link the gp17
monomers in the active multimer) appears to be unlikely 1) because this form is not immunoprecipitable and 2) because very little IgG is found
relative to gp17 in the most highly active multimeric form (fraction 1 in Figs. 6B and 7A and data not shown); however, this possibility cannot be entirely eliminated.
The proposal that terminase large subunits can assume different
conformational states is not a novel one (10, 21, 33, 34, 39). More
novel is the possibility that gp17 and other large subunit terminase
proteins may belong to a class of recently described
intrinsically unstructured proteins (40, 41). Some of the properties of
gp17 suggest that it fits criteria for inclusion in this class; thus,
gp17 1) is implicated in a critical regulatory stage of phage
development (DNA packaging); 2) interacts with multiple proteins,
including gp16, gp20, gp32, and gp55 (17) and presumably DNA; 3) binds
and hydrolyzes ATP; 4) is highly susceptible to proteolysis; and 5)
exists in multiple interconvertible conformational states. The gp16
small subunit interaction is most important for conversion to a highly
active and catalytic form, but other protein interactions may be
necessary to lock the protein into specific functional conformations
for packaging.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phage, gp18 and gp19 in phage T7/T3, gp2 and gp3 in phage
P22, and gp3 and gp16 in phage
29.) As generally found among these
phage terminases, the T4 small subunit confers the specific
DNA-binding/association properties (1-3). The large subunit contains
the prohead-binding and putative DNA-translocating ATPase activities
(3-7). However, because the measured in vitro ATPase
activities of isolated terminase proteins (8, 9) have been low in
comparison with measured rates of ATP consumption in packaging (10,
11), the identification of terminases as DNA-translocating ATPases has
been problematic. What is clear is that packaging is ATP-driven and
involves the cooperation of the DNA-associated terminase proteins with
the prohead-associated portal vertex protein (7, 12, 13) in phage T4, gp20.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, successfully making the
gp17-intein/chitin-binding domain (CBD) fusion, and full-length active
gp17 was purified. To improve protein yield, a new T7 promoter-driven
plasmid from New England Biolabs Inc. (pTYB2, 7283 bp) was employed.
The gene 17 fragment was isolated from vector pH5 after
NdeI/SacII digestion and ligated into
NdeI/SacII-cut pTYB2 to make plasmid pT5 (~9074
bp). This vector was electroporated into E. coli
HMS174(DE3) and used to perform large-scale purification of active
soluble gp17 for these studies.
-D-thiogalactopyranoside, and pelleted (~340
g). ~100 g were minced with a minimum volume (200 ml) of Q buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1 mM EDTA, 5 mM MgCl2, 0.2 mM ZnCl2, and 1 mM ATP containing
10% glycerol), and the cells were stirred gently at 4 °C until
completely homogenized. The cells were French-pressed two times at
18,000 p.s.i. and spun at 14,000 × g for 20 min, and the supernatant was removed and recentrifuged overnight at
20,000 × g. The supernatant was removed, applied through Nalgene glass-fiber syringe prefilters, and loaded onto a
300-ml chitin column pre-equilibrated in Q buffer. The column was
washed with 1-2 volumes of Q buffer, 2-3 column volumes of 1 M NaCl and 0.5% Triton X-100 in Q buffer, and 1 column
volume of Q buffer. 1 column volume of Q buffer with 40 mM
dithiothreitol (DTT) was added, and the column was incubated overnight
at 4 °C prior to eluting in 9-ml fractions with Q buffer with 40 mM DTT. The fractions showed that gp17 was cleaved from the
column and eluted to near homogeneity. Peak fractions were combined,
concentrated, and re-equilibrated in Q buffer with no DTT. The final
volume was kept large to avoid precipitation of gp17, which we found to
occur at protein concentrations over ~0.75 mg/ml. The protein was
applied to a 3-ml ssDNA/dsDNA-cellulose column (Invitrogen), and the
flow-through fraction of the gp17 protein was collected. What bound to
the column was eluted in Q buffer, 1 M NaCl, and 1% Triton
X-100 and analyzed by SDS-PAGE and Western blotting. Flow-through
fractions were applied (1 ml/min) to a 5-ml blue Sepharose column
(Amersham Biosciences) equilibrated in Q buffer. After gp17 binding,
the column was washed with 25 ml of Q buffer containing 0.5 M NaCl, 1 M NaCl, 10 mM ATP, and 50 mM ATP at a flow rate of 2 ml/min. The protein was eluted
from the column in 25 ml of 50 mM ATP and 1 M
NaCl in Q buffer and dialyzed overnight against 1 liter of Q buffer
(without NaCl and ATP) and then 1 liter of Q buffer containing 30%
glycerol, the long-term storage buffer for enzyme at
20 °C. The
enzyme has been stable for >3 years under these conditions.
-32P]ATP label. Reactions were incubated at 30 °C,
and 1-µl aliquots were removed at various times and processed as
described (26).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside. For protein
preparation, cells expressing the gp17 fusion protein were induced for
3 h, centrifuged, and lysed by French press, and the clarified
supernatant containing the fusion protein was loaded onto a chitin
column (see "Experimental Procedures"). As Fig. 1B
shows, many proteins were removed in the insoluble pellet fractions
(P1 and P2 lanes) or
did not bind and eluted in the flow-through fraction of the column
(FT lanes). Many other proteins, bound nonspecifically to
chitin or associated weakly with the gp17-intein/CBD fusion protein,
could also be removed after washing the column (W1
and W2 lanes). After equilibration of the
column overnight in DTT, cleaved gp17 protein eluted over the span of
many fractions (fractions 6-36) (Fig. 1B). Further analysis
showed that the isolated protein could be concentrated from these
dilute fractions using Amicon filtration membranes, but had a
solubility limit of ~0.75 mg/ml.
View larger version (71K):
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Fig. 1.
9% SDS-PAGE and anti-gp17 antibody Western
blot analysis of pT5 cells expressing the gp17-intein/CBD fusion
protein. A, pure gp17 protein is shown with cells
before induction (I) and after 0.5, 1.5, or 3 h of
induction. M lanes, colored molecular mass markers shown in
kilodaltons. B, shown is a 9% SDS-polyacrylamide gel of
chitin column purification of gp17. P1 and
P2, removed pellet fractions; FT, chitin
flow-through fractions; W1 and
W2, 1 M NaCl and 0.5% Triton X-100
column washes; 1-36, fractions eluted after overnight
incubation in 40 mM DTT.
View larger version (34K):
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Fig. 2.
10% SDS-PAGE and anti-gp17 Western blot
analysis of purified gp17 (sample A) and gp17 after
chitin chromatography and storage for 1 week (sample
A) or 2 months (sample B) at 4 °C.
M lane, molecular mass markers shown in kilodaltons. Below
the gels, the boldface regions of the gp17 sequence
highlight the results of the N-terminal sequence analysis in confirming
the purified gp17 protein by its N-terminal sequence (MEQPIN) in sample
A and identifying two additional cleavage fragments of the
protein at its C-terminal end present in sample B (SEVFSK,
cleavage after Ala571; and EYVPVS, cleavage after
Ala598).
20 °C
or frozen at
80 °C. This purification procedure yielded ~60 mg
of gp17 from 33 liters of cells, a significant improvement over
previously described methods.
rIIA(H88) mutant phage
that produce all components necessary to assemble phage except for
terminase proteins. Addition of terminase proteins (gp16 + gp17)
in vitro to these extracts showed that new phage can be
packaged and completed, indicating that the gp17 protein is functional
(Table I). Mixed extracts of
mutant phage-infected bacteria also produced phage, as expected; but
addition of high concentrations of purified terminase proteins enhanced
activity ~10-fold. The use of this purified gp17 protein resulted in
a marked improvement (>100-fold) over titers using previously purified terminase proteins (4), approaching the wild-type level (yield of
~10%) of phage that were synthesized under the extract growth conditions and agreeing with more recently determined values (5).
In vitro packaging of infected extract concalameric DNAs
/HindIII DNA
fragments; 25-bp, 100-bp, and 1-kb DNA marker fragments; supercoiled
plasmid DNA containing genes 16 and 17; relaxed plasmid DNA cut
with blunt or sticky end restriction endonucleases; ssDNA; yeast tRNA;
and mammalian RNA samples. None showed any substantial effects on the
rates of ATP hydrolysis. Mg2+ ions were essential for the
ATPase, and EDTA abolished activity. The ATPase activity was maximal at
~5 mM MgCl2. The PEG and MgCl2 effects on ATPase are in good agreement with the data for optimal T4
in vitro packaging by addition of these components (4).
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Fig. 3.
Effect of gp16 addition on the ATPase
hydrolysis rate of gp17. In A, conditions were 1.8 µM gp17, 1 mM ATP, and 1.5% PEG. In
B, conditions were 0.7 µM gp17, 1 mM ATP, and 1.5% PEG. In C, the effect of
varying ATP concentrations on the initial rates of hydrolysis for gp17
is shown. Initial rate data are for two different gp16 concentrations
using 0.8 µM gp17. The results shown in C are
averages of three independent experiments for each point.
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Fig. 4.
Effect of anti-gp17 antiserum on the gp17
ATPase hydrolysis rate. A, addition of anti-gp17
antiserum stimulates the gp16-dependent gp17 ATPase.
Conditions were 0.8 µM gp17, 5.5 µM gp16,
and 3 µl of anti-gp17 antiserum (17AS) added.
B, purified anti-gp17 antiserum-containing IgG
(17AS) stimulates the terminase complex ATPase
(T), but serum devoid of IgG after protein A passage
(17AS( IgG)) has no effect on the terminase complex ATPase
and on ATPase itself. For B, conditions were 1 mM ATP; 1.7 µM gp17; 3.7 µM
gp16; and 2 µl of purified fraction/20-µl reaction for anti-gp17
antiserum, anti-gp17 antiserum-containing IgG, and serum devoid of IgG
added. C, kinetic analysis of the gp17 ATPase hydrolysis
rates with and without 1 µl of anti-gp17 antiserum-containing IgG
added per 0.44 µg of gp17. 1.9 µM gp16 and 5.5 µM gp16 (not shown) were incubated with 0.8 µM gp17 protein at varying concentrations of ATP.
Analysis in both cases showed that anti-gp17 antiserum-containing IgG
addition increased the velocity of the reaction
(Vmax), with no effect on the
Km for ATP.
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Fig. 5.
ATPase activity remaining in the supernatant
and pellet after immunoprecipitation of the gp16-gp17 complex with
anti-gp17 antiserum. Shown is the distribution of terminase gp17
ATPase (T) in the supernatant (A) compared with
the pellet (B) after immunoprecipitation of a mixture of 2.3 µM gp16 and 3.1 µM gp17 with anti-gp17
antibody. Shown in C is an anti-gp17 antibody Western blot
of protein in the pellet versus supernatant after
immunoprecipitation (I.P.) (corresponding to A
and B). M lane, molecular mass marker
shown in kilodaltons; lane 1, buffer-only control;
lane 2, protein A (Prot. A)-only control;
lanes 3-6, 0.57, 0.28, 0.14, and 0.09 µl of antiserum
(AS)/µg of gp17 (0.09 µl of antiserum ATPase data not
shown in A or B), respectively; Ab
lane, anti-gp17 antiserum only (0.02 µl); 17 lane,
0.22 µg of gp17. Shown in D is the percent gp17 protein as
determined by densitometric (Dens.) analysis.
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Fig. 6.
Sedimentation of gp16 and gp17 through
glycerol gradients as shown by Western blotting of fractions using
anti-gp16 and anti-gp17 antisera. A, gp16-gp17.
B, half of the supernatant (SUP.) after
immunoprecipitation (I.P.) of gp16-gp17 with anti-gp17
antiserum (17AS). In A and B, 6.6 µg
of gp17 and 15 µg of gp16 were used. In B, anti-gp17
antiserum (1.14 µl/µg of gp17) and 50 µl of 50% protein A were
added to perform the immunoprecipitation. The IgG arrow
indicates the position of antibody revealed upon longer exposures.
M lanes, molecular mass markers shown in kilodaltons.
C, ATPase activity profile of select fractions.
glyc., glycerol.
View larger version (68K):
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Fig. 7.
Sedimentation of terminase and terminase
large subunit complexes on glycerol gradients. 4.4 µg of gp17
were incubated with 15 µg of gp16 (A) or gp16
buffer only (B), and anti-gp17 antiserum (17AS;
6.8 µl/µg of gp17) was added before glycerol gradient separation.
In C, 5 µl of each 95-100-µl fraction were assayed for
ATPase activity (see "Experimental Procedures").
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phage terminase large subunit, gpA,
appear to undergo a slow N-terminal clipping at discrete sites even in
the presence of protease inhibitors and at low temperature (31). Parris
et al. (31) found that the ATPase and nuclease activities
were also retained in the truncated protein and reasoned that the N
terminus of the protein itself was labile without the binding of the
gpNu1 small subunit protein. The C-terminal regions of the large
subunit terminase proteins of
, T3, and T4 phage appear to be
essential for prohead portal interaction (12, 32), although the central
region of gp17 is also implicated (21). Thus, gp17 C-terminal
proteolysis may similarly reflect protein lability in a region lacking
a binding partner.
-factor
gp55, so it is possible that the terminase is loaded onto DNA through
its interaction with other DNA-bound proteins such as gp55-gp45 and
gp55-RNA polymerase (17).
phage,
whose in vitro gpA ATPase has been estimated at 65-120
ATPs/gpA/min (8, 33). The T4 terminase gp17 ATPase appears to be
DNA-independent, whereas the
terminase gpA is partially dependent
upon DNA (34, 35). Other terminase proteins such as those of the
29
and T3 systems, whose purifications and partial characterizations have
been described, display significant ATPase activity only when in the
presence of the prohead, DNA, and other packaging factors (10, 36, 37).
The T4 terminase gp17 activity is unique among other large subunit
terminase proteins being studied in that it has such a significant
activity only when stimulated by the presence of the small subunit
terminase. This could relate to the highly soluble and structured gp16
ring, which may act to promote specific gp17 multimer formation.
of 1 × 104 bp/min (33, 38) and
that ATP hydrolytic requirements reported in T3 and
29 of 2 bp/ATP
(10, 38) apply to T4, then a 12-subunit (700-900 kDa) gp17 terminase
multimer displays 94% of the required activity, even without the
expected additional ATPase stimulatory effects contributed by other T4
proteins (gp32 and gp20).
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Venigalla Rao, Alasdair Steven, Gerard Barcak, Richard Karpel, Richard Thompson, and Kim Collins for critically reading the manuscript and/or for helpful comments. We thank Dong Mei Xie for expert technical assistance.
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FOOTNOTES |
---|
* The work was supported by National Institutes of Health Grant AI11676.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, University of Maryland Medical School, 108 N. Greene St., Baltimore, MD 21201-1503. Tel.: 410-706-3510; Fax:
410-706-8297; E-mail: lblack@umaryland.edu.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M208574200
2 R. G. Baumann and L. W. Black, unpublished data.
3 M. N. Simon, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; CBD, chitin-binding domain; DTT, dithiothreitol; PEG, polyethylene glycol.
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REFERENCES |
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