(Received for publication, November 10, 1995; and in revised form, February 7, 1996)
From the
A plasmid was constructed that encodes all five subunits of the Escherichia coli -complex on a single artificially
constructed operon under the control of an inducible promoter. The
proteins
,
,
`,
, and
overproduced from
this artificial operon assemble efficiently in vivo, providing
an efficient source of homogeneous
-complex. The
subunit is
a truncated form of
that is produced by a translational
frameshift. When protein expression was induced in bacterial strains
containing the outer membrane protein T (OmpT) protease,
was
proteolyzed after lysis to a
-like protein,
, and
a peptide, C-
, corresponding to the C terminus of
.
N-terminal sequencing of C-
revealed a cleavage site between two
lysines at positions 429 and 430 of
. The deduced sequence of
is, therefore, only two amino acids shorter than
natural
. The proteolysis by OmpT was also shown directly by using
purified OmpT and
-complex in an in vitro reaction. A
-complex and a mixed
-
-complex
were purified from ompT
cells. When the
-complex proteins were overexpressed in ompT
bacteria, intact
-complex lacking
could be
purified.
DNA polymerase III holoenzyme (holoenzyme), ()the
replicative polymerase of Escherichia coli, is composed of 10
subunits,
,
,
,
,
,
,
`,
,
, and
. These proteins are organized in three subassemblies
that have been isolated from cells (for reviews see McHenry(1988, 1991)
and Kelman and O'Donnell (1995)): 1) pol III core (
,
,
and
) which provides the polymerase function; 2)
, the key
processivity factor of holoenzyme; and 3) DnaX-complex, a multiprotein
ATPase that recognizes the primer terminus and loads the
processivity factor onto DNA. Both the
and the
subunits of
the holoenzyme are products of the dnaX gene.
arises
through a translational frameshifting mechanism (McHenry et
al., 1989; Tsuchihashi and Kornberg, 1990; Blinkowa and Walker,
1990; Flower and McHenry, 1990) to produce a 47.4-kDa protein. The
full-length
translation product contains all of the
sequence plus an additional 22.6-kDa domain. The
subunit is a
component of native holoenzyme and has been isolated in a complex with
pol III, to form a dimeric polymerase, pol III` (McHenry, 1982).
Although can readily associate with the
-
` and
-
holoenzyme subunits to form a five-protein
-complex in vitro, this complex has never been isolated in
vivo, presumably because the interactions of
with
are
stronger than those with
-
` and
-
. Two recent
assignments of the stoichiometry of pol III*, a subassembly of all
holoenzyme subunits except
, agree: (pol
III)
`
(Onrust et al., 1995; Dallmann and McHenry, 1995). We have
recently shown that both the
- and
-complexes have well
defined stoichiometries of
`
and
`
,
respectively (Dallmann and McHenry, 1995).
There is some confusion
regarding the roles of the DnaX proteins and complexes. E. coli cells in which was eliminated by a mutation in the
frameshifting site are viable without a detectable impaired phenotype
(Blinkowa et al., 1993). These cells contain only
, and
therefore
-less pol III* appears to be able to function at the
replication fork. There is evidence that holoenzyme reconstituted in vitro without
, containing only the
dnaX product, behaves exactly like native holoenzyme, but that
reconstituted holoenzyme, lacking
, does not (Dallmann et al., 1995). Furthermore, assembly of
into holoenzyme
requires lengthy sequential incubations in the absence of specific
holoenzyme subunits, conditions that do not appear to be physiological,
but a
-only holoenzyme assembles spontaneously (Onrust et
al., 1995; Dallmann et al., 1995; Dallmann and McHenry,
1995). These results point to
as a key component of holoenzyme
and question the need for
.
On the other hand, and
have been found within the same holoenzyme assembly (Hawker and
McHenry, 1987), and it has been widely assumed that both
and
are components of holoenzyme. Evidence has been presented that
-, but not
-complex, is the key ATPase that serves to load
the
clamp (Xiao et al., 1995), although this view is
inconsistent with a requirement for
in the ATP
S hydrolytic
assembly of initiation complexes, observed with native holoenzyme
(Dallmann et al., 1995). The
-complex, independent of
holoenzyme, has been isolated from cells (McHenry and Kornberg, 1977;
McHenry et al., 1986; Maki and Kornberg, 1988), but a
-complex has not. Thus, even the ability of the
-complex to
stably form in vivo is in question.
In an effort to resolve
these apparent discrepancies, we have undertaken an in vivo approach to study the assembly of holoenzyme. As an initial step
in this approach, we report the in vivo assembly of the
overexpressed components of the -complex. Since the genes for
holoenzyme subunits are located in widely separated regions of the
chromosome, an artificial operon had to be assembled. We have
constructed an operon that contains the five structural genes for
-complex subunits and used it to overproduce
,
,
`,
, and
. These subunits assemble efficiently in
vivo, enabling purification of the resulting complex to
homogeneity. We also show that
-complex is particularly sensitive
to the OmpT protease, post-lysis, creating a protein only two amino
acids shorter than
that is active in loading
onto primed
DNA. The potential for this cleavage must be considered when
interpreting experiments on complexes purified from cell lysates since
the
-like proteolytic product
may easily be
confused with
in an isolated complex.
Figure 1:
Construction of the -complex
expression plasmid. Construction of the plasmid pDCT.2 encoding the
five subunits is described under ``Experimental Procedures.''
The oligonucleotides used for the PCR step or for insertion fragments
are listed in Table 1. P
and P
are the PA1/O4/O3 and tac promoter, respectively. All of the
plasmids shown, with the exception of pUHE21-2, also contain the
lacI
and
-lactamase genes.
A BamHI-BamHI fragment of pMAF205
(Carter et al., 1993a) carrying the holB gene
(encoding `) was ligated to the holA-containing BamHI- and BglII-digested vector (encoding
)
(Carter et al., 1992). The 3` end of holA in the
resulting construct, pD`D.2, was then modified by replacing a DraIII-KpnI fragment with one generated by the
polymerase chain reaction (PCR) using pD`D.2 as template and
oligonucleotide primers 2053 and 2054 (Table 1). The PCR product
was cut with DraIII and KpnI prior to insertion in
cleaved pD`D.2. The resulting plasmid, pD`DS.7, contained SpeI
and SphI cloning sites immediately downstream of the holB gene.
Initially, the dnaX gene was added to
pD`DS.7 to produce a plasmid containing holA, holB,
and dnaX, but this plasmid was unstable in strain MC1061 even
without induction by IPTG, presumably due to leaky expression through
the tac promoter (P) and toxicity of the
expressed genes. Consequently, the tac promoter in pD`DS.7 was
replaced by promoter PA1/O4/O3 (P
), an artificial
combination of early RNA polymerase-dependent T7-promoter A1 and two lac operators (Lanzer and Bujard, 1988). The P
promoter is 136-fold more repressible than the tac promoter. (
)An SspI-EcoRI fragment
from pUHE21-2 (provided by Dr. H. Bujard) containing the P
promoter was used to replace the corresponding
P
-containing fragment in pD`DS.7 resulting in plasmid
pD`DSP.4. An XbaI-SphI fragment of pRT610A (Dallmann et al., 1995), containing the dnaX gene modified in
the frameshifting site to express only
, was ligated with the SpeI- and SphI-digested vector, pD`DSP.4, to produce
pDPT.5. The P
promoter resulted in a more stable holA-holB-dnaX construct than the
corresponding tac promoter-containing plasmid, when tested in E. coli strain MC1061.
An XbaI-KpnI fragment of pDPT.5, including holA, holB, and dnaX, was ligated with a SpeI- and KpnI-digested vector, pCPM.1, containing holC and holD, to create the five-subunit-expressing plasmid pDCT.2. The vector pCPM.1 was derived from a previously published construct, pMAF310 (Carter et al., 1993b), by replacing a SalI-PstI fragment, located just downstream of holD, with complementary oligonucleotides 2311 and 2312 (Table 1), which included SpeI and KpnI cloning sites.
Gene expression in the five-subunit plasmid pDCT.2 is
regulated by the tac promoter which was derived from the
vector pCPM.1. A plasmid containing the P promoter instead
of the tac promoter was also generated. Both plasmids were
relatively stable when tested in strain MC1061, suggesting that
expression of the five-subunit complex is less toxic than expression of
just
,
`, and
. The plasmid containing the tac promoter was used for the experiments reported.
Figure 2:
SP Sepharose chromatography of
-complex expressed in MC1061. A, column profile from a
20-ml column (25-cm height
1-cm diameter) equilibrated with
buffer TBP + 20 mM NaCl and loaded with 4.9 ml of the
Fraction II dialyzed ammonium sulfate pellet. After a 2-column volume
wash, the complexes were eluted in 2.5-ml fractions with a 20 to 300
mM NaCl gradient in buffer TBP at a flow rate of 0.7-column
volume/h. Protein concentration (
), conductivity (+), and
activity (
) are shown. B, gel electrophoresis of column
fractions. Samples were electrophoresed on a 7.5-17.5% gradient
SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue.
Column fractions (25 µl each) are indicated above the gel (Fr.
No.). Lanes marked M contain
and
standards; P is the column load, predialysis (25 µg); and L is the column load, post-dialysis (25 µg). The positions of
the
-complex subunits are marked;
migrates in
the same position as
.
The proteolysis
of upon dialysis was reproducibly observed when the complex was
overexpressed in two different ompT proficient strains, MC1061
and MGC1029. The degree of proteolysis varied between different
preparations, resulting in varying ratios of protein in the
-complex peak to the protein in the
-
-complex peak. To show that the
-
-complex was produced by proteolysis before
chromatography rather than by proteolysis of an already eluted
-complex, fractions 46 to 50 from the column represented in Fig. 2were pooled, diluted to reduce ionic strength, and
rechromatographed on the column reequilibrated with starting buffer.
The resulting column profile (Fig. 3A) showed a single
peak eluting at the same position as in the original column. SDS-gel
electrophoresis of the fractions (Fig. 3B) and activity
measurements (Table 3) demonstrated that no further proteolysis
had occurred and that all components eluted in equivalent ratios across
the peak, consistent with their presence as a complex with each other.
Thus, the
subunit in the peak eluted from the first
column was part of a stable
-
-complex.
Figure 3:
SP Sepharose rechromatography of the
-
-complex peak. A, fractions 46-50
from the column shown in Fig. 2were pooled, diluted to reduce
ionic strength, and rechromatographed on the same column reequilibrated
with the starting buffer. The column was then developed as described in Fig. 2. B, gel electrophoresis of column fractions
performed as described in Fig. 2. Column fractions (40 µl
each) are indicated above the gel (Fr. No.). Lanes marked M contain
and
standards; L is the column
load (20 µg).
To prove that ompT was responsible for the observed
proteolysis, strains MGC1029 and MGC1030 were constructed that were
isogenic except for a kanamycin insertion in the ompT gene of
strain MGC1030. Following transformation with pDCT.2 and induction of
protein expression, cell lysates and ammonium sulfate precipitates were
prepared for both of these strains. The Fraction II pellets were
dissolved in buffer TBP + 20 mM NaCl and dialyzed
overnight. was proteolyzed during dialysis in Fraction II
prepared from MGC1029 (ompT
) (Fig. 4, lane 5), but was not proteolyzed when expressed in MGC1030 (ompT
) (Fig. 4, lane 7).
Figure 4:
Gel electrophoresis of -complex
overexpressed in ompT mutant strain. The
-complex was
overexpressed in either strain MC1061 (ompT
),
MGC1029 (ompT
) or MGC1030 (ompT) and
Fraction II ammonium sulfate precipitates were prepared. Fraction II
pellets were dissolved in buffer TBP + 20 mM NaCl and
dialyzed overnight. Samples (15 µg each) were electrophoresed on a
10% SDS-polyacrylamide gel and stained with Coomassie Blue. Lane
1,
and
standards; lanes 2 and 3,
pre- and post-dialysis samples, respectively, from MC1061; lanes 4 and 5, pre- and post-dialysis samples, respectively, from
MGC1029; lanes 6 and 7, pre- and post-dialysis
samples, respectively, from MGC1030.
Figure 5:
Time course of the digestion of purified
-complex with pure OmpT. Reactions were performed as described
under ``Experimental Procedures'' using
-complex
overexpressed in E. coli strain MGC1030 and purified. Aliquots
representing time points were electrophoresed on a 12%
SDS-polyacrylamide gel and stained with Coomassie Blue. Lanes
1-17 correspond to 0, 2, 4, 6, 8, 10, 12, 15, 18, 21, 30,
35, 50, 60, 90, 120, and 180 min of digestion, respectively. Lanes
18-22 correspond to preincubation of OmpT for 30, 60, 120,
and 180 min, respectively, before adding
-complex and digesting
for 10 more min.
The ammonium sulfate
pellet was dissolved in buffer 25T5 using a volume that gave a solution
conductivity equal to that of buffer T5 + 20 mM NaCl, the
SP Sepharose column equilibration buffer. The sample was
chromatographed on a 210 ml of SP Sepharose column (25-cm height
2.5-cm diameter) and eluted with a 20-300 mM NaCl gradient in buffer T5 with a flow rate of 0.42 column
volume/h. There was no
-complex peak or
complexed with
in the main peak (Fig. 6A and B). The peak fraction, fraction 71, of the major
-complex peak had a conductivity equivalent to 180 mM NaCl, comparable to 190 mM NaCl for the peak fraction of
the
-
peak shown in Fig. 2. The specific
activity of the
-complex peak, 8.6
10
units/mg, is approximately half that of the
-
-complex, 1.7
10
units/mg (Table 3).
Figure 6:
SP Sepharose chromatography of
-complex expressed in MGC1030 (ompT). A, column
profile of a 210-ml column equilibrated with buffer T5 + 20 mM NaCl and loaded with 148 ml of Fraction II. After a 2-column
volume wash, the complex was eluted in 25-ml fractions with a 20 to 300
mM NaCl gradient in buffer T5 at a flow rate of 0.4 column
volumes/h. Protein concentration (
), conductivity (+), and
activity (
) are shown. B, gel electrophoresis of column
fractions performed as described in Fig. 2. Column fractions (40
µl each) are indicated above the gel (Fr. No.). The lane
marked M contains
and
standards; L is the
column load (25 µg).
For a final purification step, fractions
70-72 of the SP Sepharose column were pooled and the protein
precipitated by adding an equal volume of saturated ammonium sulfate
and centrifuging. The pellet, containing 53 mg of protein, was
dissolved in 2.5 ml of a buffer composed of 25 mM Hepes (pH
7.5), 100 mM NaCl, and 5% glycerol, and applied to a 200-ml
Sephacryl S400 HR gel filtration column (54-cm height 2-cm
diameter) equilibrated in the same buffer. The complex eluted as a
single peak at approximately 120 ml (not shown). Some purification (9.9
10
from 8.6
10
units/mg, Table 6), a change of buffer, and elimination of any aggregates
was accomplished by this step.
We have constructed a plasmid, pDCT.2, in which the five
subunits of the E. coli -complex are linked on a single
operon under the control of the inducible tac promoter. This
plasmid has allowed us to overexpress and purify a
-complex that
was assembled in vivo. This is the first report of the
isolation of the
-complex from E. coli cell lysates, and
of the stable assembly of
-complex within the cell. It is unclear
whether a
-complex occurs naturally in wild-type E. coli,
but probably exists as a component of holoenzyme in cells that were
genetically modified to knock out
(Blinkowa et al.,
1993). During the course of a purification of pol III* from those
cells,
was progressively replaced by a
-like protein,
presumably due to proteolysis (Blinkowa et al., 1993). We have
shown in this report that OmpT is responsible for such a cleavage
during purification of an overexpressed
-complex.
When we
purified the -complex which was overexpressed in ompT
strains MC1061 and MGC1029,
was
proteolyzed to a
-like protein,
(Fig. 4),
during dialysis of the dissolved ammonium sulfate pellets (Fraction
II). The proteolysis does not occur without dialysis; a nondialyzed
aliquot kept overnight on ice did not exhibit proteolysis. It is
possible that the cleavage was induced by a conformational change in
the
-complex that occurred when the ionic strength was reduced
during dialysis or that the protease activity is inhibited by moderate
salt concentration. Since the cleavage did not occur when the complex
was purified from strain MGC1030, which is isogenic with MGC1029 except
for ompT, the major protease was identified. OmpT, located in
the outer membrane of E. coli, has been purified (Sugimura and
Nishihara, 1988), but its in vivo function is not known. There
may be minor proteases that also cleave
at or very near the OmpT
cleavage site, but whose effect is not seen on our overproduced
complex. However, such hypothetical proteases may cleave a significant
portion of non-overproduced
-complex in an ompT strain.
The exact cleavage site was determined by N-terminal amino acid
sequencing of the peptide product, C-, corresponding to the C
terminus of
(Table 5). The site is between two lysines at
positions 429 and 430 of the 643 amino acids in
. OmpT is known to
have a substrate specificity for paired basic residues (Grodberg and
Dunn, 1988; Sugimura and Higashi, 1988). This site is probably near the
end of a structural domain; there is evidence that OmpT acts on
relatively unstructured regions of substrates (White et al.,
1995).
The deduced sequence at the C terminus of is . . . Lys-Ala-Lys (position 429), which can be compared to
that at the C terminus of authentic
: . . . Lys-Ala-Lys-Lys-Glu
(position 431). Since these proteins differ by only two amino acids,
they are not easily distinguished by gel electrophoresis or functional
activity. Thus, care must be taken in assigning the
isolated in
complexes without further characterization. Additional experiments are
needed to clarify the issues our findings raise relative to the
composition of complexes that contain
.
The measured
stoichiometry of -complex,
(
`)
,
is not surprising in view of the previously determined values for the
-complex of
(
`)
(Dallmann and McHenry, 1995). The result obtained for the
-
-complex,
(
`)
,
is consistent with a mixture of
`
and
`
. The actual
composition of this mixture presumably varies depending on the extent
of proteolysis. The degree of proteolysis during the dialysis step of
the purification varied, possibly due to differences in the length of
dialysis and the amount of protease in the ammonium sulfate
precipitate. Aliquots of the dialyzed protein kept at 4 °C for
several days revealed more extensive cleavage and some secondary
cleavage sites were observed. We have also obtained
-
-complex from preparations that contain very low
levels of
-complex. This may be indicative of an
asymmetry within the
tetramer of the
-complex,
where two
s are more susceptible to cleavage, although it is
unclear whether this putative asymmetry is due to the binding of
,
`,
, and
or is intrinsic to the
tetramer.
Although has been isolated in a complex with
,
`,
, and
(Maki and Kornberg, 1988), a
-complex or a mixed
-
-complex has never been isolated
from wild type E. coli. This is probably a consequence of the
isolation procedure and the fact that
but not
has a strong
affinity for polymerase core. Pol III* is treated with 1 M NaCl to dissociate the
-complex, leaving Pol III` behind;
this same treatment probably strips the
,
`,
, and
subunits from
. Alternatively, a
`
complex may
have been proteolyzed to
`
in
previous isolation attempts from cells.
Digestion of purified
-complex with purified OmpT revealed the same proteolysis
products,
and C-
, that were observed during the
purification of
-complex from ompT
strains. The same in vitro digestion products were
observed whether the
-complex substrate was assembled in vivo or reconstituted in vitro from purified components. Thus,
OmpT acts directly on the
-complex rather than indirectly through
cleavage of an intermediate. The time course further showed that the
amount of
is progressively reduced and presumably all of it can
eventually be proteolyzed.
Two other products, designated L and S (Fig. 5), were formed by cleavage at secondary sites. The L
product, migrating at approximately 56 kDa, was transient; its
concentration decreased after about 15 min. This product was never
observed upon proteolysis during purification of the overexpressed
complex (see Fig. 2B and Fig. 4), perhaps
because of its transient nature. A good candidate for the L product is
the larger of the fragments produced by cleavage after the basic amino
acid at position 133. The S product, approximately 17.6 kDa, was
observed in the Fraction II experiments ( Fig. 4and data not
shown), especially in those exhibiting extensive proteolysis.
Inspection of the sequence revealed paired basic residues at
positions 472 and 473 and at 628 and 629, among others, out of 643
amino acids. Cleavage at both of these potential sites would produce
the observed 17.6-kDa S product. Interestingly, the other
-complex
subunits are not subject to digestion, suggesting a protected and
structured environment for these proteins.
Purification of the
-complex from strain MC1061 by SP Sepharose chromatography yielded
a
-complex and a mixed
-
-complex
due to proteolysis. Rechromatography of the apparent
-
complex demonstrated that the
subunit was part
of a stable complex. This ruled out the possibility that a
-complex was eluted from the column and subsequently cleaved to
form a
product that was not part of the complex. This
is significant because the association of
with
in a complex
is difficult to achieve by in vitro assembly (Dallmann et
al., 1995; Dallmann and McHenry, 1995; Onrust et al.,
1995). Purified
and
exist as oligomers (Dallmann and
McHenry, 1995). Experiments with an overproducing plasmid that
expresses both
and
revealed no detectable quantities of
mixed oligomers formed in vivo, although it has been reported
that a
-
mixed complex can be formed in vitro by
driving the equilibrium with excess
(Onrust et al.,
1995). In vitro assembly of pol III* or holoenzyme by mixing
all 10 subunits and gel filtration produces a
-less complex
(Dallmann and McHenry, 1995; Onrust et al., 1995). The
formation of a
-containing pol III* does not occur when intact pol
III` and the
-complex are used, although a method for
reconstituting
into holoenzyme that requires an ordered addition
of subunits and an excess of
in the mix has been reported (Onrust et al., 1995). Any
assembly containing
will not
bind
, and any
assembly containing
will not bind
(Onrust et al., 1995). Together, these results suggest a
complex in vivo mechanism of
entry into holoenzyme.
Further studies with even more complex artificial operons will be
required to determine whether barriers present in in vitro assembly reactions are overcome in vivo.