(Received for publication, June 2, 1995; and in revised form, August 23, 1995)
From the
The alternative forms of the DnaX protein found in Escherichia coli DNA polymerase III holoenzyme, and
, were purified from extracts of strains carrying overexpressing
plasmids mutated in their frameshifting sequences such that they
produced only one subunit or the other. The purified subunits were used
to reconstitute the
and
complexes which were characterized
by functional assays. The
complex-reconstituted holoenzyme
required a stoichiometric excess of DNA polymerase III core, beyond
physiological levels, for activity. The
subunit stimulated the
complex 2-fold, but could not be used to reconstitute a
holoenzyme with
complex and stoichiometric quantities of core. In
the presence of adenosine 5`-O-(3`-thiotriphospate)
(ATP
S), the DNA polymerase III holoenzyme behaves as an asymmetric
dimer; it can form only initiation complexes with primed DNA in
one-half of the enzyme (Johanson, K. O., and McHenry, C. S.(1984) J. Biol. Chem. 259, 4589-4595). An asymmetric
distribution of two products of the dnaX gene,
and
, has been postulated to underlie the asymmetry of holoenzyme. To
provide a direct test for this hypothesis, we reconstituted holoenzyme
containing only the
or
DnaX proteins. We observed that,
although
could function in the presence of ATP and high
concentrations of DNA polymerase III core, it was nearly inert in the
presence of ATP
S. In contrast,
-containing holoenzyme behaved
exactly like native holoenzyme in the presence of ATP
S. These
results implicate
as a key component required to reconstitute
holoenzyme with native behavior and show that
plays a key role in
initiation complex formation. These results also show that
is not
a necessary component, since all of the known properties of native
holoenzyme can be reproduced with a 9-subunit
-holoenzyme.
The DNA polymerase III holoenzyme ()of Escherichia coli can serve as a prototype for replicative
complexes in all cells. Like many other complex mechanisms of
macromolecular synthesis, the fundamental mechanisms of DNA replication
are conserved throughout biology. The chemistry and direction of
synthesis, the requirement for RNA primers, the mechanisms of
semi-discontinuous replication with Okazaki fragments on the lagging
strand, and the need for well defined origins are shared. There are
also striking similarities between the individual components of the
complex machinery responsible for this process.
The replicative
polymerase assumes its special role because of its ability to interact
with other specialized proteins at the replication fork. In E.
coli, the DNA polymerase III core (-
-
) interacts
with a
subunit sliding clamp to enable maximum processivity
(LaDuca et al., 1986). The gene 45 protein of T4 and
eukaryotic PCNA exhibit similar properties (Burgers and Yoder, 1993;
Reddy et al., 1993). The sliding clamp in all replication
systems is a bracelet-like multimeric protein clasped around DNA. It
contacts the polymerase, providing a tether to increase processivity.
The crystal structures of yeast PCNA and E. coli
are
nearly superimposable (Kong et al., 1992; Krishna et
al., 1994). Functional and structural studies of the T4 gene 45
sliding clamp indicate that it forms similar structures and functions
like
and PCNA (Hacker and Alberts, 1994; Kaboord and Benkovic,
1993; Gogol et al., 1992; Venkatesan and Nossal, 1982).
In E. coli, the sliding clamp is set by either of two DnaX
complexes that contain either of two products of the dnaX
gene,
or
, in a complex with
-
` and
-
(McHenry et al., 1986; Maki and Kornberg, 1988;
O'Donnell and Studwell, 1990). The activity of this complex
requires the ATPase activity of the DnaX proteins, presumably to couple
ATP hydrolysis to
assembly on primed DNA (Lee and Walker, 1987;
Hawker and McHenry, 1987; O'Donnell et al. 1993,
Oberfelder and McHenry, 1987). Two additional proteins,
and
`, are required to assemble a functional clamp loader (Onrust and
O'Donnell, 1993). The
-
proteins are also
participants; they bind to DnaX and increase its affinity for
-
` so that they can cooperatively assemble a functional
complex at physiological protein levels (Olson et al., 1995).
In eukaryotes, a 5-protein complex (Activator 1, RFC) is responsible
for transferring the sliding clamp onto a primer terminus in an
ATP-dependent reaction (Lee et al., 1991; Bunz et
al., 1993). The proteins exhibit sequence homology between
eukaryotes and both Gram-negative and -positive prokaryotes, and would
be expected to act by a similar mechanism (Carter et al.,
1993; O'Donnell et al., 1993).
Asymmetric replicative
complexes have been proposed in both eukaryotic and prokaryotic
systems. In eukaryotes the polymerase has been suggested as the
leading strand polymerase and
as the lagging strand (Turchi and
Bambara, 1993; Araki et al., 1992;
Hübscher and Thömmes, 1992;
Burgers, 1991; Nethanel and Kaufmann, 1990). In E. coli, the
replicative complex apparently forms a dimer (McHenry, 1982;
Studwell-Vaughan and O'Donnell, 1991) that behaves asymmetrically
(Johanson and McHenry, 1984; McHenry and Johanson, 1984), and proposals
have been made concerning the enzyme having a distinguishable leading
and lagging strand, capable of performing the distinct specialized
functions expected of each polymerase (Hawker and McHenry, 1987;
McHenry, 1988; Maki et al., 1988). The initial observation
that provided functional evidence for asymmetry and led to the
formulation of the asymmetric dimer hypothesis came from the use of the
ATP analog ATP
S in initiation complex formation in native
holoenzyme in a concerted reaction where the DnaX complex assembled the
clamp in the presence of associated polymerase (Johanson and
McHenry, 1984; McHenry and Johanson, 1984). All of the enzyme formed
initiation complexes in the presence of ATP, but only one-half of the
enzyme entered functioning initiation complexes in the presence of
ATP
S (Johanson and McHenry, 1984). This asymmetry in the presence
of ATP
S was not an equilibrium artifact, since the 50/50
distribution was independent of ATP
S concentration once the enzyme
was saturated, and the reactions did not progress beyond the 50% level
with increasing time. It was proposed that this asymmetric behavior
arose not from two distinct populations of enzymes in solution, but
from a difference between two halves of an asymmetric dimeric DNA
polymerase with distinguishable leading and lagging strand halves.
Efforts to determine the basis of this functional asymmetry have
focused on the two products of the dnaX gene, and
(Hawker and McHenry, 1987; McHenry, 1988; Maki et al., 1988;
O'Donnell and Studwell, 1990). Translation of dnaX mRNA
produces full-length
(71,000 Da) and a product resulting from
-1 translational frameshifting into a frame with a stop codon,
(47,400 Da) (McHenry et al., 1989; Tsuchihashi and
Kornberg, 1990; Blinkowa and Walker, 1990; Flower and McHenry, 1990).
These two products were found within the same holoenzyme assembly
(Hawker and McHenry, 1987) and it was suggested that an asymmetric
holoenzyme may arise from an asymmetric assembly of these two DnaX
proteins (Hawker and McHenry, 1987; McHenry, 1988; Maki et
al., 1988). Consistent with this hypothesis,
has been shown
to influence synthesis on the two strands asymmetrically on natural
coupled replication forks (Wu et al., 1992).
Despite the
attractive hypothesis concerning asymmetric placement of and
within holoenzyme, a definitive test has not been made. In this
report, we describe our use of a DnaX overproducing strain engineered
to produce only
or
. This enabled purification of
,
free of possible proteolytic breakdown products of
, and provided
a rich source of the
subunit, free of possible contaminating
. We used these purified subunits to explore the functional
differences in their contribution to the holoenzyme reaction and to
definitively test whether DnaX protein placement underlies the
functional asymmetry of holoenzyme.
Figure 1:
Construction of
overexpression plasmids that produce only or
. A,
plasmid map of plasmid pRT610. Plasmid pRT610 was constructed as
described under ``Experimental Procedures,'' and expresses
both the
and
subunits upon induction with
isopropyl-
-D-thiogalactoside. B, site-directed
mutagenesis of dnaX. Plasmid pRT610 was mutagenized using
oligonucleotides 2 and 3 to produce plasmids that expressed only
or
, as described under ``Experimental Procedures'' and
``Results.''
To provide a plasmid
suitable for production of single-stranded DNA for mutagenesis, we
cloned the fl phage origin (obtained as a HincII fragment from
pDM1) into the NaeI site of PBBMD11 resulting in plasmid
pRT610. Plasmid pRT610 was transformed into strain XL-1 Blue (recA1, endA1, gyrA96, thi, hsdR17 (rk,mk+), supE44, relA1,
, lac
, {F`, proAB, lacI
Z
M15, Tn10(tet
)},
Stratagene, La Jolla, CA) to obtain single-stranded DNA.
Oligonucleotide-directed in vitro mutagenesis of pRT610 was
performed as described by Kunkel (1985) and Kunkel et
al.(1987). The resulting plasmids were sequenced to verify the
incorporation of the proper mutations.
Fraction I was precipitated with 107 g of ammonium sulfate (0.226 g
for each ml of fraction I, 40% saturation) and centrifuged at 22,000
g for 30 min. Pellets were backwashed by resuspension
in a Dounce homogenizer with 100 ml of buffer TBP + 0.1 M NaCl containing 0.2 g/ml ammonium sulfate (35% saturation), and
recentrifuged as before. A second backwash using 50 ml of 0.17 g/ml
ammonium sulfate (30% saturation) in buffer TBP + 0.1 M NaCl was done as above. The final pellets were flash frozen in
liquid nitrogen and stored frozen at -80 °C as fraction II.
The plasmid
producing only , pRT610A, was created using oligonucleotide 2 (Fig. 1B), which eliminated the frameshift region by
altering two codons in the sequence, AAA AAG, to AAG AAA, without
altering the corresponding Lys-Lys in the protein. The G
A
mutation stabilized an A-U interaction between the mutant mRNA and the
anticodon, perhaps discouraging slippage, but more importantly, the A
G mutation eliminated frameshifting by placing a G residue at a
nonwobble position in the -1 frame where G-U base pairs are not
possible (Flower and McHenry, 1990). This construct allowed the
expression of
only. The plasmid producing only
, pRT610B,
was created using oligonucleotide 3 for mutagenesis. This included the
same mutations as in the
-overproducing plasmid, eliminating
frameshifting, but also included an in-frame codon for glutamate,
followed by a stop codon, allowing expression of a protein equivalent
to authentic frameshifted
.
Figure 2:
Purification of . Samples of
fractions I to IV were denatured in SDS sample buffer and subjected to
electrophoresis on a 12% SDS-polyacrylamide gel. The gel was stained in
Coomassie Brilliant Blue G-250. Lane 1, fraction I (cell
lysate); lane 2, fraction II (ammonium sulfate); lane
3, fraction III (SP-Sepharose peak); lane 4, fraction IV
(S-400 HR peak). All lanes contain 30,000 units of
(equivalent to
5 µg of fraction IV).
Figure 3:
Purification of . Samples of
Fractions I to IV were boiled in SDS sample buffer and subjected to
electrophoresis on a 12% polyacrylamide gel. The gel was stained with
Coomassie Brilliant Blue G-250. Lane 1, fraction I (cell
lysate); lane 2, fraction II (ammonium sulfate); lane
3, fraction III (Q-Sepharose peak); lane 4, fraction IV
(Sephacryl S-300 HR peak). All lanes contain 80,000 units of
(equivalent to 5 µg of fraction IV).
Preliminary experiments in which and
were
purified from a strain containing the expression plasmid pBBMD11, which
contained the wild-type dnaX sequence and co-expressed both
and
, indicated that mixed
/
oligomers do not form
to a significant extent in the absence of other overproduced subunits in vivo. Fig. 4A shows the protein and DNA
synthesis activity profile of fraction II prepared from a strain
containing pBBMD11 chromatographed on an S-Sepharose ion exchange
column. Gel electrophoresis of the two major peaks in the profile
revealed only
in the first peak, while the second peak contained
only
(Fig. 4B). A mixed
/
oligomer would be
expected to elute at a position between pure
and
; however,
no such peak was seen. This observation implies that
and
do
not form mixed oligomers in vivo when co-expressed, consistent
with the results of Tsuchihashi and Kornberg(1989), who also purified
and
from a co-expressing plasmid and observed no mixed
oligomers of
and
.
Figure 4:
Co-expression of and
subunits
does not yield mixed complexes.
and
were overexpressed from
plasmid pBBMD11. Fractions I and II were prepared as described under
``Experimental Procedures.'' A, S-Sepharose
chromatography. Fraction II was chromatographed over a 100-ml
S-Sepharose (Pharmacia) column equilibrated in buffer TBP.
and
were eluted with a 10-column volume gradient of 0-300
mM NaCl in buffer TBP. Protein concentration (
),
conductivity (
), and activity (
) are shown. B,
gel electrophoresis of S-Sepharose column fractions. Samples of
fractions I and II and selected column fractions were boiled in SDS
sample buffer and subjected to electrophoresis on a 12% polyacrylamide
gel. The gel was stained with Coomassie Brilliant Blue G-250. Lane
1, molecular weight markers; lane 2, fraction I (cell
lysate), 10 µg; lane 3, fraction II (ammonium sulfate), 5
µg; lanes 3-20, column fractions 50, 53, 56, 59, 62,
65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, and 97 (volume equivalent
to 0.5 µl of each column fraction).
Initiation
complexes were formed using ATP or ATPS with
complex, core,
and
(Fig. 5A), with
complex, high
concentrations of core, and
(Fig. 5B) and with
native holoenzyme purified from E. coli (Fig. 5C). Using native holoenzyme, ATP
S
resulted in approximately one-half the amount of initiation complexes
formed compared to ATP, in agreement with the results of Johanson and
McHenry (1984). As expected, ATP also supported initiation complex
formation with both
and
complexes, whereas ATP
S did
not support initiation complex formation with
complex and only
supported half the amount of initiation complex formation with
complex, compared to ATP. The result with
complex-reconstituted
holoenzyme was identical to that observed with native holoenzyme (Fig. 5C) (Johanson and McHenry, 1984). Thus, the
subunit/complex must play a major role in loading
subunit clamps
onto primed DNA in native holoenzyme. A striking result from this
experiment was that
complex-reconstituted holoenzyme was
indistinguishable from native holoenzyme, whereas
complex-reconstituted holoenzyme was very different. Clearly the
asymmetric behavior of holoenzyme, as revealed by the differential
effects of ATP and ATP
S, was not due to asymmetric placement of
and
within holoenzyme, but instead reflects properties of
the
subunit alone.
Figure 5:
Differential effects of ATPS on
and
complexes. A, effect of ATP
S on initiation
complex formation by
complex.
complex (100 fmol), core (240
fmol), and
(400 fmol) were assayed for the ability to form
initiation complexes on primed, SSB-coated M13G
(540 pmol
as nucleotide) as a function of ATP (
) or ATP
S (
)
concentration as described under ``Experimental Procedures.'' B, effect of ATP
S on initiation complex formation by
complex.
complex (100 fmol), core (4 pmol), and
(400
fmol) were assayed for the ability to form initiation complexes on
primed, SSB-coated M13G
(540 pmol as nucleotide) as a
function of ATP (
) or ATP
S (
) concentration as
described under ``Experimental Procedures.'' Note that a high
level of polymerase III core was used to fully enable the catalytic
potential of
. Lower activity was observed with the low polymerase
III core levels used in A. C, effect of ATP
S on
initiation complex formation by native DNA polymerase III holoenzyme.
70 units of DNA polymerase III holoenzyme were assayed for the ability
to form initiation complexes on primed, SSB-coated M13G
(540 pmol as nucleotide) as a function of ATP (
) or
ATP
S (
) concentration as described under ``Experimental
Procedures.''
To exclude the possibility that the above
result reflected partial proteolysis of the subunit to a
-like protein (Blinkova et al. 1993), an experiment was
carried out in which initiation complexes prepared using
complex
with ATP and ATP
S were purified by gel filtration over Bio-Gel A5m
(Bio-Rad) columns (Johanson and McHenry, 1984), and fractions were
analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting
using an anti-
/
polyclonal antibody. No cleavage of
to
a
-like protein was observed. Furthermore, activity assays of the
column fractions showed that only half the amount of initiation complex
was obtained with ATP
S compared to ATP (data not shown),
consistent with the above results.
Figure 6:
Large excess of core is required for
complex activity. The
complex (100 fmol) (
) and
complex (100 fmol) (
) were mixed with the indicated amounts of
core,
(400 fmol), SSB-coated M13G
(540 pmol as
nucleotide), and ATP (10 µM). A control experiment
(
) containing only core and
was also performed. All
remaining assay components were added and incubated at 30 °C for 5
min as described under ``Experimental
Procedures.''
Figure 7:
Titration of the subunit into
and
complex assays. The
complex (100 fmol) was assayed with
200 fmol of core (
) and the indicated amounts of
. The
complex (100 fmol) was assayed with 200 fmol (
), 400 fmol
(
), and 1 pmol (
) of core and the indicated amounts of
. Each assay contained 400 fmol of
, 540 pmol of SSB-coated
M13G
(as nucleotide), and 10 µM ATP. All
remaining assay components were mixed and incubated at 30 °C for 5
min as described under ``Experimental
Procedures.''
The
2-fold stimulation of complex by
might also be explained by
a reequilibration of the
complex subunits during assays and the
formation of significant amounts of
complex in situ.
However, when the
subunit was preincubated with the
complex
at 15 °C for various times before assaying, no time-dependent
increase in activity occurred (Fig. 8). A control assay in which
no
was added gave 39 pmol of DNA synthesis. All other assays
showed a 2-fold stimulation independent of preincubation time. This
result suggests that any reequilibration of
complex into
complex in these assays is not responsible for the observed
stimulation.
Figure 8:
Stimulation of complex by
is
independent of preincubation. The
complex (100 fmol) was
preincubated with
(200 fmol) for the indicated times at 15
°C. Each assay contained core (280 fmol),
(400 fmol),
SSB-coated M13G
(540 pmol as nucleotide), and ATP (10
µM). All remaining assay components were added and
incubated at 30 °C for 5 min as described under ``Experimental
Procedures.'' A control assay in which no
subunit was added
yielded 39 pmol of DNA synthesis, approximately 2-fold lower than the
data shown, consistent with the results shown in Fig. 7.
The and
subunits of DNA polymerase III holoenzyme
were purified from overexpressing plasmids which produce only the
or
subunits (Fig. 1Fig. 2Fig. 3; Table 1and Table 2) in order to investigate the functions
and physical properties of each subunit alone, without interference
from the other. Experiments with an overproducing plasmid that
co-expressed both
and
from the wild-type dnaX gene
showed that the
and
subunits, overexpressed by themselves,
do not form detectable quantities of mixed oligomers in vivo (Fig. 4), consistent with the observation of Tsuchihashi
and Kornberg (1989). It has been suggested (Stukenberg et al.,
1994; O'Donnell, 1994) that a mixed
assembly can be
formed in vitro by incubation of
with
in the
absence of other holoenzme subunits. However, our observation that
and
do not form detectable mixed assemblies in vivo shows that this assembly is not favored, at least not in the
absence of other overproduced proteins.
The purified and
subunits were used to reconstitute the
complex
(
`
) and
complex
(
`
) (Dallmann and McHenry, 1995). These
complexes were then mixed with polymerase III core and the
subunit in order to test the hypothesis that an asymmetric distribution
of
and
in holoenzyme (Hawker and McHenry, 1987) was
responsible for the asymmetric activity of holoenzyme when assayed with
ATP
S (Fig. 5C) (Johanson and McHenry, 1984). In
these experiments, ATP
S supported only half the amount of
initiation complex formation as ATP. A possible interpretation is that
only one of the DnaX components, either
or
, can hydrolyze
ATP
S to form initiation complexes, reflecting their asymmetric
placement in holoenzyme. The prediction for the reconstitution
experiment was that one of the
- or
-reconstituted
holoenzymes would be inactive with ATP
S and the other would
display the same activity with ATP
S as with ATP. Consistent with
this prediction, ATP
S did not support initiation complex formation
with
complex-reconstituted holoenzyme; however, with
complex-reconstituted holoenzyme, ATP
S supported only half the
amount of initiation complex formation, compared to ATP, i.e. the
complex reconstituted holoenzyme behaved exactly as
native holoenzyme. This clearly indicated that the asymmetric behavior
of holoenzyme, as revealed by the effect of ATP
S, was not due to
an asymmetric placement of
and
within holoenzyme, but
reflected properties of the
subunit alone. This result also
demonstrated that the
subunit/complex plays a role in loading
subunit clamps onto primed DNA in native holoenzyme, a point that
was not previously clear.
The above results raised a number of
questions about the role of in holoenzyme. The
subunit also
functions to dimerize core (McHenry, 1982; Studwell-Vaughan and
O'Donnell, 1991) and might serve as an organizing center for
holoenzyme, recruiting the
complex into holoenzyme (Stukenberg et al., 1994; O'Donnell, 1994). The data in Fig. 6appeared to support this suggestion, since, in the absence
of
,
complex required large amounts of core to yield
activities comparable to that of
complex with stoichiometric
amounts of core. To address this possibility, we titrated
into
complex assays at various ratios of
complex to core to
determine whether
can efficiently recruit
complex into a
holoenzyme that could function at stoichiometric levels of core (Fig. 7). Although
caused a 2-fold stimulation of
complex activity, this level of stimulation was seen at all
concentrations of core used. This observation indicated that
did
not promote more efficient recruitment of
complex into holoenzyme
over the 5-min time course of the assay, a time sufficient for the
synthesis of
300 Okazaki fragments. If it had, the stimulation by
would have been expected to be more dramatic at lower enzyme
concentrations, due to the greater effective concentration achieved
because of proximity effects as
complex was recruited into a
holoenzyme complex. The possibility that dissociation of
,
`,
and
from the
complex, and assembly of significant
amounts of
complex accounted for the results in Fig. 7was
ruled out in an experiment in which the
subunit and the
complex were preincubated (Fig. 8). The lack of any increase
over the 2-fold stimulation seen previously indicated the absence of
any stripping of
,
`, and
from
complex by
the
subunit within the time frame of the experiment.
Additionally, the half-life for the dissociation of DnaX complexes was
1.4 h at 20 °C (Olson et al., 1995), making the
possibility of reequilibration of subunits unlikely in a 5-min assay.
Holoenzyme reconstituted with the complex as the sole
clamp loader is functionally impaired when compared to native
holoenzyme or to holoenzyme reconstitued with
complex. In 100
mM potassium glutamate, a salt concentration that approximates
the lower end of physiological ionic strength, the
complex
requires core at concentrations of
160 nM in order to
achieve DNA synthesis levels approaching those of
complex
reconstituted with
10 nM core. If an E. coli cell contains 10-20 copies of holoenzyme (McHenry and
Kornberg, 1977), its intracellular concentration would be
14-28 nM based on a cell volume of
1 fl
(Ingraham et al., 1983). Clearly
complex only appears to
function at nonphysiological concentrations of core, whereas
complex exhibits maximal activity at physiological concentrations. This
study has provided a functional explanation for the observations of
Blinkova et al.(1993) who found that mutant E. coli strains which do not express
are viable, but that a
temperature-sensitive mutation which interferes with
function is
lethal. Their additional observation of rapid proteolysis of
to a
-like protein, plus the functional aspects of
activity
presented here, raise the possibility that the
subunit seen in
the physical holoenzyme complex could result from cleavage of
. Of
course, the
complex may have another ancillary role in DNA
replication or repair. For example, mismatch repair requires a product
of the dnaX gene (Lahue, et al. 1989) and would not
be expected to require assembly of a dimeric replication fork. It is
also possible that
complex could serve a role for unloading
sliding clamps left behind during holoenzyme cycling (Stukenberg et
al., 1994).
After this manuscript was under review, a paper by
O'Donnell and colleagues (Onrust et al., 1995) appeared
reporting a -
mixed complex requiring an excess of
;
significant complex formation did not occur at a 1:1 ratio between
and
, even at high, nonphysiological protein concentrations.
We interpret this as further evidence that
-
and
-
interactions are favored over mixed DnaX protein assemblies in the
absence of other holoenzyme subunits. In the absence of other
interactions that pull equilibria favoring mixed DnaX assemblies, these
would not be expected to form at significant levels in vivo.
However, our finding does not exclude more complex mechanisms involving
other holoenzyme or cellular proteins not present at adequate levels in
the DnaX-overproducing cells to redirect the assembly pathway.
In
another report in the same series (Xiao et al., 1995),
holoenzyme assembled with ATPase mutants of exhibited diminished
replication activity, while enzyme assembled with ATPase mutants of
appeared fully active. Our results with ATP
S show that the
full activity of holoenzyme can be reconstituted with
only and
that
by itself is ineffective. The most direct interpretation of
these results would be that
serves as an ATPase to reproduce
ATP
S-dependent initiation complex formation with holoenzyme.
Alternatively,
could alter the conformation of
within
holoenzyme, enabling it to use ATP
S as an effective substrate. We
infer that
can also participate in the ATPase-coupled initiation
complex formation of native holoenzyme.