©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
In Vivo Assembly of the -Complex of the DNA Polymerase III Holoenzyme Expressed from a Five-Gene Artificial Operon
CLEAVAGE OF THE -COMPLEX TO FORM A MIXED --COMPLEX BY THE OmpT PROTEASE (*)

(Received for publication, November 10, 1995; and in revised form, February 7, 1996)

Arthur E. Pritchard H. Garry Dallmann (§) Charles S. McHenry (¶)

From the Department of Biochemistry, Biophysics, and Genetics and Graduate Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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, (P), 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 (P) 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 (P)-complex and a mixed -(P)-complex were purified from ompT cells. When the -complex proteins were overexpressed in ompT bacteria, intact -complex lacking (P) could be purified.


INTRODUCTION

DNA polymerase III holoenzyme (holoenzyme), (^1)the replicative polymerase of Escherichia coli, is composed of 10 subunits, alpha, , , , , , `, , , and beta. 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 (alpha, , and ) which provides the polymerase function; 2) beta, the key processivity factor of holoenzyme; and 3) DnaX-complex, a multiprotein ATPase that recognizes the primer terminus and loads the beta 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 alpha are stronger than those with -` and -. Two recent assignments of the stoichiometry of pol III*, a subassembly of all holoenzyme subunits except beta, agree: (pol III)(2)(2)(2)` (Onrust et al., 1995; Dallmann and McHenry, 1995). We have recently shown that both the - and -complexes have well defined stoichiometries of (4)(1)`(1)(1)(1) and (4)(1)`(1)(1)(1), 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 beta clamp (Xiao et al., 1995), although this view is inconsistent with a requirement for in the ATPS 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 beta 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 (P) may easily be confused with in an isolated complex.


EXPERIMENTAL PROCEDURES

Strains and Plasmids

E. coli strain DH5alpha (F80dlacZDeltaM15 Delta(lacZYA-argF) U169 endA1 recA1 hsdR17(r(K) mK) deoR thi-1 supE44 gyrA96 relA1) was used for routine plasmid transformation and purification. Once characterized, selected plasmids were transformed into E. coli MC1061 (FhsdR mcrB araD139 Delta(araABC-leu)769 DeltalacX74 galU galK rpsL thi ), BL21 (FompT hsdS(B)(r(B)m(B)) gal dcm), MGC1029 (mcrA mcrB IN(rrnD-rrnE)1 lexA3 Delta(uvrD)::T(c)^r), or MGC1030 (mcrA mcrB IN(rrnD-rrnE)1 lexA3 Delta(uvrD)::T(c)^r Delta(ompT)::K(m)^r) for protein overproduction. MGC1029 and 1030 were derived from strain W3110 and are similar to our laboratory strain MGC1020 which is used for purification of DNA polymerase III holoenzyme (Cull and McHenry, 1995). MGC1030 was made by Pl bacteriophage transduction of the deletion-insertion (ompT)::K(m)^r from strain AD202 (Akiyama and Ito, 1990). For protein expression, E. coli strains containing plasmid were grown, induced with IPTG, and harvested using a 250-liter fermentor (New Brunswick) as described by Olson et al.(1995).

Construction of the -Complex Plasmid

Fig. 1summarizes the construction of a plasmid that encodes the five subunits (, , `, , and ) of the -complex in a single operon under the control of an inducible promoter. The strategy was to link individual subunit genes from previously constructed expression plasmids, with 20-30 nucleotides between the stop codon of the upstream gene and the Shine-Dalgarno sequence of the next gene. In the previous construction of the single-subunit overexpression plasmids, the initial codons of the genes were often changed to high usage codons synonymous with wild-type codons. These same sequences were used in the present constructs.


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^q and beta-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. (^2)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.

Buffers

Buffer TBP is 50 mM Tris (pH 7.5), 20% (w/v) glycerol, 5 mM dithiothreitol, 5 mM benzamidine, and 0.5 mM phenylmethylsulfonyl fluoride. Buffer T5 is 50 mM Tris (pH 7.5), 5% (w/v) glycerol. Buffer 25T5 is 25 mM Tris (pH 7.5), 5% (w/v) glycerol.

Preparation of Cell Lysate and Ammonium Sulfate Precipitation

The lysates (Fraction I) and ammonium sulfate precipitates (Fraction II) were prepared as described by Cull and McHenry(1995) with the following exceptions: 1) the lysis mixture was adjusted to 5 mM EDTA before lysozyme was added, 2) 0.194 g of ammonium sulfate for each ml was added to the lysate supernatant, and 3) a single ammonium sulfate backwash was performed by resuspending the initial pellet in 0.125 times Fraction I volume of backwash solution (0.15 g of ammonium sulfate added to each milliliter of buffer). These conditions were empirically determined to maximize purification without significant loss of activity.

SP-Sepharose Chromatography

Dissolved Fraction II pellets were applied to columns equilibrated with buffer T5 plus 20 mM NaCl or buffer TBP plus 20 mM NaCl as indicated. The column was washed with 2 column volumes of the indicated equilibration buffer and developed using a 9-column volume gradient of 20 to 300 mM NaCl in the buffer T5 or TBP at a flow rate of 0.7 column volume/h.

DNA Polymerization Assays

Activity of the -complex was measured by reconstituting holoenzyme and measuring DNA synthesis from a primed M13Gori template. Assays contained, in 25 µl, 573 fmol of beta, 500 pmol of M13Gori (as nucleotide), 165 units (40 ng) of dnaG primase, 1.6 µg of E. coli single-stranded DNA binding protein, 1 pmol of pol III core (alpha), and 0.6 to 6 fmol (adjusted to be in the linear range of the assay) of DnaX complex. Reactions were performed in a buffer containing 50 mM Hepes-KOH (pH 7.5), 10% (v/v) glycerol, 0.1 M potassium glutamate, 10 mM dithiothreitol, 10 mM magnesium acetate, 200 µg/ml bovine serum albumin, 0.02% (v/v) Tween 20, 10 µM ATP, 48 µM dATP, dCTP, and dGTP, and 18 µM [^3H]TTP. Assays were incubated at 30 °C for 5 min, and quenched by trichloroacetic acid precipitation. One unit is 1 pmol of total deoxyribonucleotide incorporated/min.

Protein Sequencing

Samples were adsorbed to a ProBlot membrane, washed with 20% aqueous methanol, and subjected to N-terminal sequence analysis using standard Edman chemistry by Dr. James McManaman at the University of Colorado Cancer Center Protein Chemistry Laboratory.

In Vitro Proteolysis of -Complex by OmpT Protease

The substrate was either 1) -complex reconstituted from purified components, , , `, , and , and then purified by Mono Q chromatography as described by Dallmann and McHenry(1995), or 2) -complex overproduced in E. coli strain MGC1030 and purified as described below. After SP Sepharose chromatography, the complex was further purified by gel filtration on a Sephacryl S400 HR resin (Pharmacia Biotech Inc.). Reactions were performed at 37 °C in 25 mM Hepes (pH 7.5), 50 mM NaCl, 5% glycerol, 1 mM dithiothreitol, and 0.1% Nonidet P-40. Reaction time points were obtained by withdrawing 10-µl aliquots from the reaction, mixing with 10 µl of stop buffer (0.18 M Tris (pH 6.8), 30% sucrose, 6% SDS, 180 mM dithiothreitol), and immediately incubating at 95 °C for 2 min. Each reaction aliquot contained 5 µg of -complex and 4 ng of OmpT (obtained from Patricia Babbitt, University of California, San Francisco). Control reactions to monitor stability of OmpT were carried out by incubating the reaction mixture lacking substrate at 37 °C for the indicated times, withdrawing 7.5-µl aliquots, and incubating each with 2.5 µl of -complex substrate for an additional 10 min followed by termination as described above. Reaction products were separated by SDS-polyacrylamide gel electrophoresis, stained with Coomassie Blue, and scanned using a laser densitometer as described by Dallmann and McHenry(1995).

Other Procedures

Chromatographic supports, proteins, holoenzyme subunits, and nucleic acids were obtained as described by Olson et al.(1995). SDS-polyacrylamide gel electrophoresis and protein determinations were performed as described by Olson et al.(1995). Stoichiometries of complex subunits were determined by scanning gels as described by Dallmann and McHenry (1995), except that complex constituent standards were not included on the gel. Instead, the molar ratio of each subunit relative to was derived from the ratios of the corresponding scanned band intensities by assuming that the proportionality of the molar ratio to the band intensity ratio is the same on different gels and using proportionality constants provided by the standard data generated by Dallmann and McHenry(1995).


RESULTS

Overproduction of -Complex

We constructed a plasmid with all five structural genes for the -complex linked in an artificial operon (Fig. 1). The dnaX gene was modified at the frameshifting site so that only was expressed (Dallmann et al., 1995). Induction of the operon led to overexpression of , , `, , and and efficient assembly of a functional complex as evidenced by the 50-fold increase in activity found in the lysates containing induced -complex proteins compared to lysates containing only overproduced and (Table 2). To develop a purification scheme, the ammonium sulfate precipitation step was first optimized (Table 2). Initially three precipitation conditions were used corresponding to 0.258, 0.226, and 0.194 g of ammonium sulfate added for each ml of lysate. A high specific activity, 1.6 times 10^6 units/mg, was achieved with only slight loss of total activity by using the product from the 0.194-g ammonium sulfate precipitation (Table 2, initial cut). Of two ammonium sulfate backwashes of this product, the 0.15 backwash gave a specific activity of 4.5 times 10^6 units/mg (Table 2) with no loss of total activity. This represents a large purification, since the total amount of protein in this backwash is only 2% of the total protein in the Fraction I lysate supernatant.



Cleavage of to (P)

The lysate (Fraction I) and ammonium sulfate precipitate (Fraction II) were prepared from 18.5 g of cells as described under ``Experimental Procedures.'' The Fraction II pellet was dissolved in 5 ml of buffer TBP + 20 mM NaCl and dialyzed overnight against the same buffer to reduce the conductivity. However, during dialysis, approximately two-thirds of the was proteolyzed to a product, (P), that was indistinguishable from on gels. The other half of the cleaved , denoted C-, was also detected on SDS gels (Fig. 2B). The dialyzed sample was chromatographed on an SP Sepharose column, since cation exchange chromatography gives excellent purifications for both and (Dallmann, et al., 1995). Two peaks of activity eluted with a 20-300 mM NaCl gradient in buffer TBP (Fig. 2A). SDS-polyacrylamide gel electrophoresis revealed that the peak eluting at 105 mM NaCl contained (P), , `, , and in a complex indistinguishable from authentic -complex (Fig. 2B). The (P) subunit seen in the various fractions migrates in the same position as (lane M). The second peak (190 mM NaCl) contained both (P) and , plus , `, , and . The proteolysis product, C-, eluted as a peak after the -(P)-complex peak. The specific activity of the (P)-complex, 4.1 times 10^7 units/mg, was significantly higher than that of the -(P)-complex, 1.7 times 10^7 units/mg (Table 3).


Figure 2: SP Sepharose chromatography of -complex expressed in MC1061. A, column profile from a 20-ml column (25-cm height times 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 (box), 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; (P) 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 (P)-complex peak to the protein in the -(P)-complex peak. To show that the -(P)-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 (P) subunit in the peak eluted from the first column was part of a stable -(P)-complex.


Figure 3: SP Sepharose rechromatography of the -(P)-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).



Stoichiometries of the DnaX Complexes

The gel shown in Fig. 2B was scanned using a laser densitometer and the molar ratio of each subunit relative to was determined for the peak fractions of the (P)-complex and the -(P)-complex (Table 4). The stoichiometry of the (P)-complex was (`)(1)(1), in agreement with the stoichiometry of legitimate -complex assembled in vitro (Dallmann and McHenry, 1995). The stoichiometry for the mixed -(P)-complex was determined to be (`)(1), indicating preservation of the tetramer of DnaX, but cleavage of at least half of the to (P) that remained in the complex.



Location of the Proteolysis Cleavage Site

Fig. 2B (lanes P and L) suggests proteolysis of produces the products (P) and C-. The C- product elutes from the SP Sepharose column just after the -(P)-complex peak, along with and small amounts of and . To precisely determine the cleavage site, protein from a column fraction corresponding to fraction 56 in Fig. 3was subjected to 10 cycles of N-terminal sequence analysis using standard Edman chemistry. Two predominant amino acids in approximately equal amounts were obtained with each cycle (Table 5). These results are consistent with the sequences of two proteins: 1) the C-terminal portion of starting with residue 430 of the 643 amino acid protein, and 2) the subunit. These data locate the cleavage site between positions 429 and 430 of . (^3)



Proteolysis Is Due to OmpT

Since the proteolysis cleavage site was determined to be between two lysines (amino acids 429 and 430), the outer membrane protease OmpT was considered a likely candidate in inducing the cleavage (Grodberg and Dunn, 1988; Sugimura and Higashi, 1988). The -complex-expressing plasmid, pDCT.2, was transformed into E. coli strain BL21 which has an ompT mutation. Protein overexpressed in this strain was purified through the lysate and ammonium sulfate precipitation procedures described above. Dialysis of this material did not result in any proteolysis of the subunit (results not shown).

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.



Proteolysis of -Complex by OmpT in Vitro

To verify that OmpT was directly responsible for the observed proteolysis, we performed the reaction in vitro using purified -complex and OmpT. -Complex was incubated with OmpT, and aliquots were taken at the indicated times and subjected to SDS-polyacrylamide gel electrophoresis (Fig. 5). Two sources of -complex were used: 1) -complex that had been overexpressed in E. coli strain MGC1030 (ompT) and purified through SP Sepharose and gel filtration chromatography; and 2) -complex reconstituted in vitro from purified components (Dallmann and McHenry, 1995). Within 2 min of digestion, the proteolysis products, (P) and C-, were evident. With increasing digestion time, the amount of decreased and at 3 h was less than 10% of its original amount. The amount of (P) increased rapidly and plateaued at approximately 30 min. Additional cleavage products, labeled L and S in Fig. 5, were also observed. The amount of L increased for approximately 15 min and then decreased. The amount of S increased monotonically with time. The same products seen with the -complex assembled in vivo (Fig. 5) were also observed with the reconstituted complex (data not shown). No significant loss of OmpT activity occurred in the course of the reaction, as shown by the results of reactions in which protease was incubated at 37 °C for various times without the -complex substrate, and then incubated with substrate for 10 min (Fig. 5, lanes 18-22).


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.



Purification of -Complex Overexpressed in MGC1030

Since purification of the -complex overexpressed by E. coli strain MC1061 ultimately yielded a -(P) mixed complex, we purified the complex from MGC1030 cells which lack the OmpT protease. Starting with 120 g of cells, 10 g of protein were found in the lysate which yielded 274 mg after ammonium sulfate precipitation and backwash (Table 6).



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 times 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 (P)-complex peak or (P) 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 -(P) peak shown in Fig. 2. The specific activity of the -complex peak, 8.6 times 10^6 units/mg, is approximately half that of the -(P)-complex, 1.7 times 10^7 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 (box), 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 times 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 times 10^6 from 8.6 times 10^6 units/mg, Table 6), a change of buffer, and elimination of any aggregates was accomplished by this step.


DISCUSSION

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, (P) (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 (P) 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 (P)-complex, (`)(1)(1), is not surprising in view of the previously determined values for the -complex of (`)(1)(1) (Dallmann and McHenry, 1995). The result obtained for the -(P)-complex, (`)(1), is consistent with a mixture of (2)(2)` and (1)(3)`. 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 -(P)-complex from preparations that contain very low levels of (P)-complex. This may be indicative of an asymmetry within the (4) 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 (4) 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 (2)(2)` complex may have been proteolyzed to (4)` in previous isolation attempts from cells.

Digestion of purified -complex with purified OmpT revealed the same proteolysis products, (P) 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 (P)-complex and a mixed -(P)-complex due to proteolysis. Rechromatography of the apparent -(P) complex demonstrated that the (P) 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 (P) 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.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Research Grant GM35695 and facilities support from the Lucille P. Markey Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Postdoctoral Fellowship from the Natural Sciences and Engineering Research Council of Canada.

To whom correspondence should be addressed.

(^1)
The abbreviations used are: holoenzyme, DNA polymerase III holoenzyme; OmpT, outer membrane protein T; Pol, polymerase; IPTG, isopropyl-beta-D-thiogalactoside; P, P promoter; P, tac promoter; PCR, polymerase chain reaction.

(^2)
D. R. Kim and C. S. McHenry, submitted for publication.

(^3)
The cleavage site has also been confirmed by sizing the (P) subunit using mass spectrometry. An experimentally determined mass of 47,163 agrees with a calculated value of 47,145 for a (P) produced by cleavage between amino acids 429 and 430 (A. E. Pritchard, T. Farmer, R. Caprioli, and C. S. McHenry, unpublished experiment).


ACKNOWLEDGEMENTS

We thank Dr. Patricia Clement Babbitt for the gift of OmpT protease and Millard Cull for the construction of E. coli strains MGC1029 and MGC1030.


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