(Received for publication, October 7, 1994; and in revised form, June 7, 1995)
From the
The terminase holoenzyme of bacteriophage is a
multifunctional protein composed of two subunits, gpNu1 and gpA. In
vitro, under certain conditions, terminase can render DNAs from
various sources, of varying lengths and termini, resistant to
degradation by high concentrations of DNase I. This reaction is
completely dependent on the presence of terminase, proheads, a
hydrolyzable triphosphate, and a divalent metal ion, and we propose
that it is the result of translocation of DNA into proheads by
terminase. This reaction is stoichiometric with respect to terminase,
DNA, and proheads and can be supported by all deoxyribo- and
ribonucleoside triphosphates, but not by the corresponding diphosphates
or nonhydrolyzable ATP analogs. Mg
and Ca
promote the reaction, but Mn
and Zn
do not. In the absence of spermidine, translocase activity is
low, but addition of the Escherichia coli protein integration
host factor (IHF) promotes specific translocation of only those DNA
fragments containing the terminase-binding site, cosB. When
spermidine is present, nonspecific translocation of DNA from any source
is stimulated. Under these conditions IHF no longer promotes
specificity, but translocation of only cosB-containing DNA
fragments can be restored by addition of small amounts of a dialyzed
and RNase-treated E. coli extract, suggesting that additional
host factor(s) may be involved in determination of packaging
specificity. To a limited extent, gpA alone can promote translocation,
but gpNu1, which has no translocase activity on its own, must be added
to approach the holoenzyme-like activity levels. Formation of viable
phage cannot be accomplished by gpA in the absence of gpNu1.
During the assembly of phage heads in vivo,
linear DNA concatamers are matured by the terminase holoenzyme and then
specifically translocated into the preformed protein shells, or
proheads, starting from the left end of the genome (reviewed in Feiss
(1986); Becker and Murialdo(1990); Murialdo(1991)). This translocation
reaction is believed to be accomplished by a similar process in all
double-stranded DNA bacteriophages (Casjens, 1985; Black, 1988) and is
thought to be driven by ATP hydrolysis, since an ATP requirement exists
for all in vitro packaging systems (Earnshaw and Casjens,
1980; Black, 1988). Another essential requirement for DNA packaging is
the presence of the functional terminase proteins, which are thought to
be directly involved in the translocation process (Becker et al., 1977; Guo et al., 1986; Hamada et al., 1986; Rao
and Black, 1988). Specificity of
DNA maturation and translocation in vivo derives from the interactions of the terminase
holoenzyme with the DNA elements of the cohesive end junction (cos site), located between -40 and +160 bp on the
genome map (Miwa and Matsubara, 1982, 1983; Hohn, 1983; Feiss et
al., 1983). Cos contains three distinct regions: cosN (-11 to +11 bp), (
)a sequence with partial
2-fold rotational symmetry where terminase makes the staggered nicks
and separates the 12-base overhangs using an ATP hydrolysis-requiring
helicase-like activity (Higgins et al., 1988; Rubinchik et
al., 1994b); cosB (+12 to +160 bp), which
contains three binding sites, R1, R2, and R3, to which both the small
subunit of terminase, gpNu1, and the terminase holoenzyme, specifically
bind (Shinder and Gold, 1988; Parris et al., 1994), as well as
a high affinity binding site for IHF (Kosturko et al., 1989);
and cosQ (-12 to -40 bp), a region involved in
completion of DNA translocation into the prohead and the cleavage of
the 2nd cos site (Cue and Feiss, 1993).
It is clear that
the two subunits of terminase, gpA (74 kDa) and gpNu1 (21 kDa),
must contain domains which define the numerous activities performed by
terminase as part of the overall packaging pathway. We have recently
established that both the ATPase and the helicase activities of the
terminase holoenzyme can be accounted for by its large subunit, gpA
(Parris et al., 1994), and have quantitatively analyzed the
reaction parameters of these activities (Rubinchik et al.,
1994b). We have attempted to investigate the relationship between the
two known ATP hydrolysis-requiring activities of terminase, packaging
and helicase, and the observed in vitro ATPase. Our
interpretation of the results suggested that the in vitro ATPase of gpA and terminase is predominately associated with the
helicase activity of these proteins and not necessarily with their
translocation activity. However, this interpretation was questionable
since the roles and requirements of the various host proteins, as well
as of phage morphogenic components required to complete the phage
assembly pathway and generate viable PFUs, were not defined in the in vitro packaging assay. DNA packaging assays measure the
formation of viable, infectious phage particles, and this formation
consists of a series of steps. After the
terminase
DNA
prohead complex is formed, DNA is translocated
into the capsid. Thereafter, the phage proteins gpD, gpW, and gpFII
must be added before tails can be attached (Perucchetti et
al., 1988), and there is evidence that host factor(s) may also be
involved. In this study, we have used a defined in vitro assay
to look specifically at the translocation of DNA into a preformed
prohead, in the absence of additional host or phage components, and to
quantitatively describe the translocase activity promoted by
terminase and its subunits.
For
the in vitro assays, all proteins were first dialyzed against
buffer containing 10 mM Tris-HCl, pH 8.0, 1 mM EDTA,
and 10% (v/v) glycerol. Protein concentrations were determined after
dialysis, using the Bio-Rad protein assay, according to the
manufacturer's instructions. Terminase was assumed to have the M of 120,000. Deoxyribonuclease I (DNase I, 1861
units/mg dry weight) was obtained from Worthington Biochemical
Corporation. Pyruvate kinase (2 mg/ml) was obtained from Boehringer
Mannheim. HinDIII, EcoRI, PstI, and ScaI restriction endonucleases and T4 ligase were obtained
from New England Biolabs. Acetylated bovine serum albumin was obtained
from New England Biolabs as a 10 mg/ml stock solution.
The reactions were initiated by the addition of DNA and were typically incubated at room temperature for 60 min. Unless stated otherwise, 1 µl of a DNase I solution (10 mg/ml) was then added to each reaction, and incubation continued for an additional 10 min. The reactions were stopped by the addition of 3-6 µl of gel loading buffer, which contained 60% glycerol, 150 mM EDTA, and 0.05% bromphenol blue. The reactions were then either electrophoresed directly, or packaged DNA was released from proheads by adding 2-4 µl of 10% SDS to the reactions and incubating them for 5 min at 65 °C. Electrophoresis was carried out in 0.7% agarose gels in 0.04 M Tris acetate and 0.002 M EDTA buffer, pH 7.7, at 12 V/cm for 1-2 h. Subsequently, gels were either stained in 0.01% ethidium bromide solution and photographed, or, if labeled DNA was used, dried and exposed to storage phosphor plates overnight, and scanned by the PhosphorImager instrument from Pharmacia LKB. Dried agarose gels could also be stained with Coomassie Brilliant Blue R for protein analysis. Quantitative data analysis was performed by the GraFit data analysis application (version 2.04, Erithacus Software Ltd.), as described previously (Rubinchik et al., 1994a, 1994b). Specifically, all above-background counts remaining in a lane (including the well) after the DNase I treatment were considered to be the result of translocation-dependent protection.
DNase I sensitivity assays were performed with the following modifications to the above system. After the assembly of phase I reactions (with or without gpD and gpW, as desired), they were incubated for 15 min to allow packaging of the DNA to occur. At this point 2 µg of DNase I were added and reactions left to proceed for another 10 min at room temperature. Appropriate phase II components were added and the reaction incubated for another hour at room temperature. Viable phage were titered as described above.
DNA packaging assays with other DNA substrates to
test for packaging specificity and infectivity of translocated DNA were
performed as the 2-phase assays described above, except that DNA
was replaced by T7 or supercoiled, linear, or concatameric plasmid DNA
of the same concentration. T7 DNA packaging was tested by plating phase
II products on QD5003 and DH5 and screening for the appearance of
characteristic T7 plaques. Plasmid infectivity was monitored by
incubation of a plasmid DNA packaging reaction aliquot (10 µl) with
0.1 ml of an exponential culture of QD5003 in 2 ml of culture broth for
1 h at 37 °C, followed by plating on solid agar plates containing
ampicillin (50 µg/ml), and by observing growth of
ampicillin-resistant colonies.
Figure 1:
In vitro packaging reactions result in formation of DNase I-resistant DNA.
The in vitro packaging assays were carried out in 15-µl
reaction volumes and under HS conditions, treated with DNase I,
electrophoresed, and scanned by PhosphorImager as described under
``Experimental Procedures.'' All reactions contained 0.5
µg of mature DNA, of which 0.1 µg was
C-labeled and 150 nM terminase. Lanes 1 and 14, untreated [
C]
DNA; lanes 2 and 13, 1 µl of cesium chloride
gradient-purified
phage containing
C-labeled DNA; lane 3, 5 µl of 594 sonicate; lane 4, 5 µl of
NS428 sonicate; lane 5, 5 µl of
594(
Eam
cI
Sam
)
sonicate; lane 6, 5 µl of
594(
Bam
cI
Sam
)
sonicate; lane 7, 5 µl of
594(
Cam
cI
Sam
)
sonicate; lane 8, 5 µl of
594(
Nu3am
cI
Sam
)
sonicate; lane 9, 5 µl of
594(
Aam
Dam
cI
Sam
)
sonicate; lane 10, 5 µl of
594(
Aam
Wam
cI
Sam
)
sonicate; lane 11, 5 µl of
594(
Aam
Kam
cI
Sam
)
sonicate; lane 12, 5 µl of 594 sonicate plus 1.5 µg of
purified proheads. Sonicates were prepared from induced lysogens as
described under ``Experimental
Procedures.''
Figure 2:
Purified proheads and terminase are both
necessary and sufficient to promote DNase I resistance. The translocase
assays were carried out in 10-µl reaction volumes and under HS
conditions, as described under ``Experimental Procedures,''
except that DNase I was not added to lanes 1, 3, 5, and 7 after a 60-min incubation. All reactions
contained 0.8 µg mature DNA, of which 0.1 µg was
C-labeled. Lanes 3, 4, 7, and 8 contained 150 nM terminase; lanes 5-8 contained 9 nM purified
proheads.
Heating the
DNase I-treated reactions at 65 °C in the presence of 1.5% SDS
released the DNA from proheads, which then appeared as a distinct band
with a mobility similar to that of mature DNA. Due to the limited
resolution of DNA fragments larger than 20 kb on agarose gels, we
estimate the size of DNA species recovered from the proheads to be
between 25 and 48.5 kb. The last estimate, which corresponds to the
complete length of the
genome, is unlikely to be accurate, since
the products of the translocation assay did not generate viable PFUs
after DNase I treatment, unless complemented with partially purified
gpD and gpW (Table 2).
Figure 3:
Effect of reaction conditions on
specificity of translocation by terminase. All reactions contained 150
nM terminase, 8 nM proheads, and 0.3 µg of mature
DNA, digested with HinDIII. Lanes 2, 4, 6, 8, and 10 contained 30
nM IHF. Reaction conditions were: lanes 1 and 2, RS; lanes 3 and 4, RS + 50 mM NaCl; lanes 5 and 6, RS + 3 mM spermidine; lanes 7 and 8, HS; lanes 9 and 10, HS + 15 mM putrescine. Lane 11 contained 0.2 µg of
HinDIII digest as a
control. After DNase I treatment, translocation reactions were heated
at 65 °C in the presence of SDS, as described under
``Experimental Procedures.''
Spermidine, or a
combination of spermidine and putrescine, significantly enhanced the
nonspecific translocase activity, so that under HS conditions all
linear DNA molecules of varying lengths and termini, such as all the
fragments of a HinDIII digest, monomeric T7 genomes, or
plasmid DNA, could be translocated into
proheads, regardless of
the presence of the cos site (Fig. 3, lanes 5, 7, and 9; Table 3). Likewise, eukaryotic DNA,
from yeast and calf thymus, was translocated under these conditions.
However, only those DNA molecules that contained both the left and the
right ends of
chromosome, such as mature
chromosomes
themselves, or concatameric cosmid DNA, (
)gave rise to
infectious particles, as monitored by the appearance of plaques and
ampicillin-resistant colonies, respectively (Table 3).
Specificity of translocation in the presence of spermidine or both
spermidine and putrescine could not be restored by IHF (Fig. 3, lanes 6-8), but was accomplished by addition of small
amounts of dialyzed E. coli extract treated with RNase A (Fig. 4, lanes 3 and 5).
Figure 4:
Substrate specificity of the terminase in vitro translocase activity is modified by unidentified host
factor(s). Lane 1, 0.1 µg of mature C-labeled
DNA, digested with HinDIII, and heated at 65 °C. Lanes 2-5, translocase assays containing 150 nM terminase, 8 nM proheads, and 0.3 µg of HinDIII-digested mature
C-labeled
DNA. Lanes 3 and 5, reactions were heated at 65 °C in
the presence of SDS following the DNase I treatment. Lanes 2 and 3, HS reaction conditions; lanes 4 and 5, HS conditions + 1 µl of dialyzed and RNase-treated E. coli C sonicate.
Figure 5:
Protein concentration dependence of the
translocase activity of terminase, gpA, and gpA:gpNu1. Data points
represent means and standard deviations of the 60-min reaction yields
from at least two experiments. All reactions contained 8 nM proheads and 3 nM mature DNA, of which 0.6 nM was
C-labeled. Reaction conditions were RS +
IHF.
, terminase;
, gpNu1 and gpA, in a 3:1 molar ratio,
with protein concentrations on the graph being that of gpA;
,
gpA.
Figure 6:
Kinetics of the translocase activity of
terminase in the presence of IHF. , 17 nM terminase;
, 50 nM terminase. Reaction volumes were scaled up
6-fold. Other reaction conditions were as described in Fig. 4and under ``Experimental Procedures.'' 10-µl
aliquots were removed at the indicated times, and the extent of
translocation was determined. Data points represent means and standard
deviations of three independent experiments. Data points for each time
course were fitted with the curves according to the first order rate
equation.
The translocase activity was also found to be stoichiometric with respect to the reaction concentrations of DNA (Fig. 7A) and proheads (Fig. 7B). That is, the yield was directly proportional to the amount of the component tested up to the point where it saturated one of the other reaction components (Fig. 7B). We propose that the number of translocation-competent complexes formed is determined by the reaction concentrations of terminase, DNA, and proheads, as well as relevant physiological conditions, such as spermidine and IHF.
Figure 7:
Proheads and DNA concentration dependence
of the translocase activity of terminase. Reactions contained 150
nM of terminase and either 8 nM proheads and
different concentrations of mature DNA (A), or 4 nM mature
DNA and different concentrations of proheads (B). Reaction conditions were RS + IHF. Data points
represent means and standard deviations of the 60 min reaction yields
from three independent experiments.
The
translocase activity of terminase was optimal at MgCl concentrations of between 1 and 5 mM. It could also be
supported by similar concentrations of CaCl
, but not by
MnCl
or ZnCl
. Translocase was inactive if ATP
was absent from the reaction, or if it was replaced by ADP, or by
nonhydrolyzable analogs such as ATP
S and
,
-methylene-ATP. The activity could also be supported, to a
varying extent, by all NTPs and dNTPs, with ATP and dATP being the most
efficient, followed closely by CTP. We have investigated the effect of
varying the ATP concentration on the translocation reaction (Fig. 8). 50% of maximal activity was reached at ATP
concentrations of approximately 60 µM, but when
concentrations higher than 1 mM were present the activity
decreased sharply. This decrease was determined to be the result of
Mg
depletion by the nucleotide and could be reversed
by increasing the concentration of MgCl
.
Figure 8:
Dependence of the translocase activity of
terminase on the concentration of ATP. The 15 min yields of the
packaging reactions were plotted as a percent of maximum activity
against the reaction concentration of ATP. Data points represent means
and standard deviations from two independent experiments. , 150
nM terminase. Reaction conditions were RS + IHF, as
described under ``Experimental Procedures.'' To maintain
initial ATP concentrations, phosphoenol pyruvate and pyruvate kinase
were added to the reactions to the final concentrations of 10 mM and 5 µg/ml, respectively. Approximate ATP concentration at
which 50% of activity was reached is
indicated.
Under RS plus IHF reaction conditions, combining gpA and gpNu1 produced translocase activity comparable to that of the terminase holoenzyme, albeit somewhat less active on a molar basis (Fig. 5). Activity profiles with respect to substrate specificity, as well as various metals, nucleotides, and inhibitors, were also essentially the same. The optimal subunit ratio was between 2 and 4 gpNu1 molecules to 1 gpA molecule (Fig. 9). The subunit mixture was inactive under HS conditions.
Figure 9: The effect of varying the gpNu1/gpA molar ratio on the translocase activity of gpA. The concentration of gpA was held constant at 600 nM. Varying amounts of purified gpNu1 were mixed with gpA, and the mixture was kept on ice for 15 min prior to the addition of other reaction components. Data points represent means and standard deviations of 60-min reaction yields of three independent experiments. All reaction conditions were RS + IHF. Assay analysis was as described under ``Experimental Procedures.''
Bacteriophage in vitro packaging assays are
very inefficient, with less than 0.05% of the substrate DNA generating
PFUs in our experiments (Table 2). By using
C-labeled
DNA as a substrate in these assays, we
were able to observe some of it becoming resistant to DNase I
degradation. This resistance did not require phage proteins responsible
for the maturation of
heads (gpD, gpW, or gpFII), or the presence
of phage tails (Fig. 1). DNase I-resistant DNA migrated in a
novel pattern on an agarose gel, presumably as the result of its
association with phage morphogenic components. Since this resistance
required the presence of preassembled
proheads, active
terminase, hydrolyzable NTPs, and, in a purified system, could lead to
the formation of viable phage after the DNase I treatment, we believe
that it arises as the result of translocation of DNA into the
proheads. The amount of DNA protected was at least 100-fold higher than
expected from the number of PFUs observed (Table 2), suggesting
that either the viability of the large number of filled proheads was
lost at some subsequent morphogenic step (perhaps addition of the
tails), or that the majority of DNA translocated into proheads was in
some way inviable to begin with (for example, damaged ssDNA overhangs).
Translocation activity was not detected with extracts that lacked gpB
or gpNu3, which is consistent with previously reported observations
that these mutants cannot synthesize
preconnectors (Georgopoulos et al., 1983). It was also absent in gpE
extracts, which lack the prohead shell, but do make the
BC-preconnectors (Georgopoulos et al., 1983). Interestingly, a
significant amount of translocation activity was detected with
gpC
extracts (Fig. 1, Table 2), which
have been reported to make inviable prohead-like structures containing
gpE, gpB, and gpB*, and partially filled with gpNu3 (Georgopoulos et al., 1983). Therefore, the element of the connector
composed of gpB (and/or its derivative, gpB*) appears to be sufficient
for interaction with the terminase during DNA translocation.
Next,
we demonstrated that purified proheads could be used successfully
in the place of sonicates of induced lysogens. Under HS conditions,
terminase and proheads were both necessary and sufficient for the
translocase activity to occur (Fig. 2). Translocase was fully
active in the presence of RNase, making it unlikely that RNA molecules
play an important role in this process in
, as they do in
29
(Guo et al., 1987). Translocation also occurred under RS
conditions, although much less efficiently. In the presence of IHF,
however, the reaction yields under RS conditions equaled or even
surpassed those obtained under HS. Both sets of conditions could be
used to assemble mature heads in a two-stage in vitro packaging assay (Table 2), indicating that spermidine was
not essential to the translocation process itself, but probably acted
to facilitate the formation of the DNA
terminase complexes.
Presumably, IHF acts in a similar fashion, its binding to the high
affinity site in cosB enhancing gpNu1-mediated terminase
interactions with that region, as has been demonstrated in vitro for gpNu1 (Shinder and Gold, 1989). The stimulatory effect was not
cumulative, i.e. no additional stimulation occurred if IHF was
added to the translocase reactions under HS conditions. A similar
observation was made on the effects of spermidine and IHF on the in
vitro endonuclease activity of terminase (Rubinchik et
al., 1994a).
When the products of the translocase assay were
denatured in the presence of SDS, the released DNA migrated through the
0.7% agarose gel as a single band with mobility similar to that of
mature . This implies that most of the length of input DNA was
packaged into purified proheads. However, when assayed as a substrate
for subsequent production of viable phage in a two-stage in vitro packaging assay, the translocase products were found to be
sensitive to DNase I treatment (Table 2), suggesting that the
entire
genome could not be packaged into the prohead in the
absence of gpD, in agreement with published results obtained in
vivo (Sternberg and Weisberg, 1977). Adding gpD and gpW to the
translocation assay prior to the addition of DNase I did indeed render
the reaction products almost completely insensitive to degradation, as
witness the number of PFUs generated (Table 2). These
observations are consistent with the previously described roles for gpD
and gpW as proteins required to ensure the packaging of the entire
genome and the stabilization of the mature
head
(Perucchetti et al., 1988).
We have investigated the
specificity of the in vitro translocase activity of terminase
by utilizing a number of nucleic acids as substrates. Neither
supercoiled DNA, ssDNA, nor RNA molecules could be translocated under
any conditions tested (Table 3). Under RS conditions and in the
presence of IHF, the DNA molecules that contained the chromosome
left end cosB site were specifically and preferentially
translocated (Fig. 3, Table 3). Under HS conditions,
however, all linear dsDNA molecules could be translocated, irrespective
of origin, composition, or the nature of their termini. Such
promiscuous translocase activity in vitro has been previously
described in a number of other bacteriophages which utilize DNA
elements to ensure specificity of packaging in vivo, such as
T3 (Hashimoto and Fujisawa, 1988), T7 (Son and Serwer, 1992), and P22
(Behnisch and Schmieger, 1985). In the case of
translocation in vitro, the failure of non-linear DNA to serve as a
substrate implies that the main requirement is a double-stranded
terminus, which apparently can have either 5` or 3` ssDNA overhangs or
be blunt (Table 3). The presence of the proper mature
termini must therefore become essential at some step following the
initiation of DNA translocation. Experiments with T7 and plasmid DNA
have demonstrated that unlike T3,
cannot infect cells
successfully in the absence of the cos site, in agreement with
the previous reports that specific interactions of
tails and the
right end of the
chromosome are necessary for both tail
attachment and DNA ejection (Xu and Feiss, 1991) and that both mature
termini of the
chromosome are required for its circularization
following its injection into a bacterium.
For the in vitro translocation system, the loss of DNA specificity was correlated
with the presence of spermidine in the reactions (Fig. 3).
However, packaging of DNA by
in vivo is very specific,
both in the presence and absence of IHF, with no detectable amounts of
non-
DNA found in purified mature phages. Since bacterial cells
can contain high concentrations of spermidine and other polyamines,
additional factors must play a role in ensuring the specificity of
translocation. We have found that the spermidine-promoted loss of
specificity could be overcome by addition of small amounts of dialyzed
and RNase-treated E. coli C extract, suggesting that other, as
yet unidentified, host factor(s) may be involved in the process and
that it or they are likely to be protein in nature (Fig. 4). E. coli C was chosen because it was reported to lack cryptic
lambdoid prophage sequences (Murialdo, 1988), which would make it
unlikely that the effect on specificity of translocation was due to a
background expression of some phage protein.
The translocase
activity also required a hydrolyzable nucleoside triphosphate and a
divalent metal ion. All four NTPs and dNTPs could be utilized with
comparable efficiency, although ATP and CTP generated higher initial
rates of translocation. These results are consistent with the
previously characterized NTPase activity of terminase and gpA, but
differ from that of in vitro packaging, where CTP was 100-fold
less efficient than ATP, and GTP was completely inactive (Rubinchik et al., 1994b). The translocase activity of terminase reached
50% of maximum at approximately 60 µm of ATP (Fig. 8), a
concentration comparable with the K values of the helicase and ATPase activities of gpA and
terminase, but significantly different from the ATP requirement for in vitro packaging (Rubinchik et al., 1994b). Both
Mg
and Ca
supported translocation
equally well, while Zn
and Mn
were
inactive. In vitro packaging has a similar profile with
divalent metals, but there Ca
is 100-fold less
efficient than Mg
(Rubinchik et al., 1994b).
Similar results were obtained with studies of potential inhibitors,
where sodium vanadate inhibited packaging significantly more than it
did translocase at comparable concentrations. The comparison of
activity profiles of in vitro packaging and translocase with
nucleotides, metals, and inhibitors supports a previously proposed
hypothesis that there is some other step(s) in phage morphogenesis that
occurs during in vitro packaging that has a more stringent
requirement for ATP and Mg
, and is more sensitive to
inhibition, than the translocase activity (Rubinchik et al.,
1994b). This step could involve the activities of gpW, gpFII, or tails.
Previously, we have reported that purified terminase subunits
gpNu1 and gpA could be combined to generate in vitro endonuclease, ATPase, and packaging activities resembling those of
the terminase holoenzyme (Parris et al., 1994; Rubinchik et al., 1994a, 1994b). A similar result was obtained for the
translocase activity. The mixture of the two subunits was less active
than the holoenzyme on a molar basis (Fig. 5) but had
essentially the same activity parameters with respect to nucleotides,
metals, and inhibitors. Unlike terminase, a gpA-gpNu1 mixture was
inactive under HS conditions, as was previously reported for its
endonuclease activity (Rubinchik et al., 1994a). In both
cases, inactivation was correlated primarily with the presence of NaCl
in the reaction. The optimal gpNu1 to gpA molar ratio for the
translocase activity was between 3 and 4 (Fig. 9), which is
higher than the previously reported ratio for the endonuclease activity
(between 1 and 2, Rubinchik et al., 1994a). It is possible
that translocation of DNA requires a higher degree of occupancy of R
sites or another gpNu1 function not related to its DNA binding
activity. An intriguing possibility is that the number of gpNu1
molecules associated with each gpA within the terminase protomer
actually varies depending on the function being performed, with more
gpNu1 being ``recruited'' as needed. Such an arrangement
would be consistent with the non-integer gpNu1/gpA ratios that were
previously reported for purified terminase (Gold and Becker, 1983;
Tomka and Catalano, 1993a; Parris et al., 1994).
Not only the initial reaction rates, but also the final yields of the translocase activity were directly proportional to the reaction concentrations of terminase (Fig. 6, Table 4). The yields were also proportional to the reaction concentrations of DNA (Fig. 7A) and proheads (Fig. 7B). These observations are consistent with the model where ternary complexes are first assembled, the number of such complexes depending on the final concentrations of individual components, as well as the reaction conditions. Titrations of one of these components with the other two being in excess indicate that only a fraction of each of them appears to be capable of forming translocation-competent complexes. In the case of terminase, the amount of inactive protein may be less than it appears, since the number of terminase protomers required per active translocating complex is unknown and could be quite high. It is also possible that components damaged during purification inhibit the reaction by interacting with their active counterparts to form inactive complexes.
Translocation of DNA into proheads appears to proceed at
a high initial rate, which gradually slows down (Fig. 6). This
reaction rate is an average of the complex population and does not tell
us what is happening on the level of the individual translocating
complex. Since the in vitro translocation reaction does not
appear to be processive with respect to terminase (Fig. 5), it
is possible that the terminase complex is unable to dissociate from the
filled prohead on its own and requires activity of subsequent
morphogenic components, such as gpW, gpFII, or tails. We are
currently investigating these interactions.
Terminase has an in
vitro NTPase activity which is independent of the translocation
reaction (Gold and Becker, 1983; Tomka and Catalano, 1993b; Rubinchik et al., 1994b), while purified proheads have no
detectable NTPase of their own. When ATPase of the translocation
reaction was measured, it was found to be essentially the same as that
of terminase and DNA alone (Table 4). The average rate of ATP
hydrolysis was sufficient to account for the calculated average rates
of translocation, based on the assumption that 2 bp of DNA could be
translocated per ATP molecule hydrolyzed (Guo et al., 1987;
Morita et al., 1993).