(Received for publication, October 11, 1995; and in revised form, January 4, 1996)
From the
The TrfA protein encoded by the broad host range bacterial
plasmid RK2 specifically binds to eight direct repeats (iterons)
present at the plasmid replication origin to initiate DNA replication.
Purified TrfA protein is largely in the form of a dimer, and using a
dimerization test system that involves the fusion of the amino-terminal
domain of the cI repressor protein to TrfA, we show that the TrfA
protein forms dimers in vivo. Because of the high stability of
the dimer form of TrfA, the formation of heterodimers between the
wild-type and different sized TrfA proteins requires in vivo de
novo folding of the primary protein sequence or in vitro denaturation and renaturation. The results of gel mobility shift
assays using in vitro or in vivo formed heterodimers
indicated that the TrfA protein binds to the iteron DNA as a monomer.
Furthermore, when the monomeric and dimeric forms of TrfA are separated
by gel filtration chromatography, only the protein in the
chromatographic position of the monomeric form demonstrated significant
DNA binding activity. These results indicate that only the monomer form
of the TrfA protein is active for binding to the iterons at the RK2
replication origin.
The ability of bacterial plasmids to regulate their replication is critical to the stable maintenance of these extrachromosomal elements. RK2 and other plasmids of the incompatibility group IncP are distinguished by their ability to be stably maintained in a diverse group of Gram-negative bacteria (see (1) for references). Controlled replication of RK2 in these bacteria requires only two plasmid encoded elements: the origin of replication, oriV, and the initiation protein, TrfA(2, 3, 4) . All other replication functions are provided by the host bacterium. The trfA gene encodes two forms of the RK2 initiation protein. The smaller 33-kDa protein, TrfA-33, is the result of an independent in-frame translational start in the open reading frame used for the larger 44-kDa protein, TrfA-44 (5, 6, 7) . While the significance of two forms of the initiation protein may be in extending the host range of RK2, the TrfA-33 protein by itself is capable of initiating RK2 replication in a number of different Gram-negative bacteria(8, 9, 10) .
The
TrfA protein specifically binds to eight directly repeated sequences,
called iterons, found in the origin of
replication(11, 12) . The iterons are arranged in oriV in groups of five and three. The eight-iteron origin is
utilized in all Gram-negative bacteria examined(2) , although
the five-iteron origin is also functional in Escherichia
coli(13) . Two iterons outside of oriV have also
been identified(14, 15) . The iterons in RK2 were
originally defined as 17-bp ()sequences separated by a
4-6-bp spacer(16) . A later study demonstrated that
certain bases within the less conserved spacer sequence are also
required for specific binding of TrfA to the iterons(17) .
Binding by TrfA to the origin iterons is a key step for the regulation
of replication initiation. However, binding alone is not sufficient to
initiate replication, as several mutants of TrfA have been isolated
that are capable of binding to oriV in vitro and in
vivo, but which are defective for replication in a number of
bacterial hosts(18, 19) . Thus the TrfA protein by
itself must carry out other functions at the origin or host proteins
must interact with TrfA bound to oriV to form the prepriming
nucleoprotein structure required for DNA replication to begin.
Both
TrfA-33 and TrfA-44 proteins have been shown to purify as a dimer in
solution by sucrose gradient analysis (18) ()and by
chemical cross-linking(11, 18) . Most prokaryotic DNA
binding proteins that bind as dimers recognize sites that reflect the
symmetry of the proteins by having a dyad symmetry in the DNA sequence
(for review, see (20, 21, 22) ). The DNA
binding site for TrfA has a highly conserved region (the 17-bp iteron)
and a 5` less conserved region (the 4-6-bp spacer sequence).
Contacts with TrfA are made in both regions(17) . The TrfA
binding site does not posses any obvious dyad symmetry. However,
analysis of the methylation interference pattern of TrfA-33 protein
binding to a single binding site revealed putative TrfA
DNA
contacts localized in two adjacent major grooves on one face of the
DNA(17) . These results could be an indication that TrfA binds
as a dimer to DNA even if the sequence itself does not show an evident
symmetry.
The replication initiation proteins from several narrow host range iteron-containing plasmids have been studied. For plasmids P1(23) , pSC101(24) , and F(25) , the wild-type Rep protein is primarily purified as a dimer in solution, but, in each case, the monomeric form of the protein binds to the iteron sequences at their replication origins(26, 27, 28) . For pSC101 and F, the dimeric form of the Rep protein binds to inverted repeats that are related in sequence to the iterons and have a role in the autoregulation of rep gene expression (26, 27) . The purpose of the present study was to determine the form of the TrfA protein that binds to the iterons at the RK2 origin of replication. The results presented below indicate that while purified TrfA-33 protein is largely a dimer in solution and forms, at least to some extent, dimers in E. coli cells, it is the monomer form of this protein that is bound to the iterons of RK2 oriV.
For expression of the His6-TrfA fusion protein the vector pAT30 was
constructed as follows. The 0.9-kb EcoRI-PstI trfA-33 gene fragment from plasmid pRD110-16 (31) was cloned into the same sites of the pSELECT vector
(Promega Corp., Madison, WI). ()This plasmid was then
mutagenized using the oligonucleotide 5`-ACACGCGAGGAtCcATGACGACCAAG-3`
and the Altered Sites in vitro Mutagenesis System following
directions supplied by the manufacturer (Promega). The mutagenesis
altered two bases (noted in lower case above) to create a BamHI site adjacent to the start of the trfA gene
sequence coding for TrfA-33 resulting in plasmid pAT18. The 0.9-kb BamHI-PstI trfA fragment from pAT18 was then
cloned into the same sites of pTrcHis-A (Invitrogen Corp., San Diego,
CA), yielding an in-frame fusion of a 36-amino acid polypeptide with
the TrfA-33 sequence. This plasmid was designated pAT30.
It was
necessary to reclone the gene expressing His6-TrfA from pAT30 into a
vector compatible with the pBR325 based vectors used to express TrfA-33
(plasmid pBK3) (11) and TrfA-N123 (plasmid
pKK233-2-N
123)(18) . This was achieved by digesting
pAT30 with EcoRV, ligating the digestion products to EcoRI linkers, and then digesting again with EcoRI.
The 2-kb EcoRI fragment (which included the gene encoding
His6-TrfA as well as the upstream elements required for expression of
the fusion protein) was cloned into the unique EcoRI site of
the R6K-derived plasmid pRR15(32) . This resulted in plasmid
pAT35 in which expression of His6-TrfA was in the opposite direction as
the lacZ
of the vector.
His6-TrfA was purified from E. coli BL21 (pAT30) as follows. The strain was grown on R
medium (2% tryptone, 1% yeast extract, 0.5% NaCl, 0.2% glycerol, 50
mM KPO, pH 7.2) containing 200 µg/ml
penicillin at 30 °C to an A
of 1.0.
Isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 0.5 mM, and the culture was grown an
additional 2-6 h before harvesting. To prepare a cleared lysate,
the cell pellet was resuspended at 4 °C in Buffer A (50 mM NaPO
, 300 mM NaCl, 0.05% Triton X-100, pH
8.0) containing 50 µg/ml phenylmethylsulfonyl fluoride using 3 ml
of buffer for each gram wet weight of cells. The cell suspension was
lysed by sonication, and the lysate was cleared by centrifugation at
85,000
g for 30 min at 4 °C.
His6-TrfA was
purified from the lysate by affinity to a Ni-NTA resin (Qiagen Inc.,
Chatsworth, CA) using approximately 4 ml of resin for 24 ml of cleared
lysate. The cleared lysate was added to resin (prewashed with Buffer
A), and the sample was incubated at 4 °C for 30 min on a rotating
table. The lysate-resin was spun 1.5 min in a clinical centrifuge at
room temperature, and the supernatant (containing the unbound proteins)
was discarded. The protein-resin was then washed with Buffer A 3 times
using 5 times the volume of resin per wash. For each wash, the
protein-resin plus buffer were incubated for 1-5 min on a
rotating table at 4 °C and then spun for 1.5 min in a clinical
centrifuge at room temperature. The protein-resin was then washed 4
times with Buffer A containing 10% glycerol and 40 mM imidazole using 5 the volume of resin per wash. For the
first two washes, the sample was incubated for 5-10 min on a
rotating table at 4 °C before spinning. For the last two washes,
the sample was mixed briefly before spinning. The protein-resin was
next washed 2 times using 5
Buffer H
(50 mM NaPO
, 300 mM NaCl, pH 6.0) and then
resuspended in an equal volume of Buffer H
(50 mM NaPO
, 300 mM NaCl, pH 5.0). The protein-resin
was then poured into an empty column at 4 °C. After the resin had
settled, the column was washed using Buffer H
until the A
of the column effluent was less then 0.01.
His6-TrfA was then eluted using Buffer H
(50 mM NaPO
, 300 mM NaCl, pH 4.0). Peak fractions
were then pooled and dialyzed against 50 mM NaPO
,
100 mM NaCl, pH 6.0, and stored at -70 °C.
The
His6-TrfA plus TrfA-33 or the His6-TrfA plus TrfA-N123 proteins
were purified as above using E. coli BL21 (pAT35)(pBK3) or E. coli BL21 (pAT35)(pKK233-2-N
123), respectively,
grown on R medium containing 200 µg/ml penicillin and 40 µg/ml
kanamycin as the starting cultures.
A mixture of 100 µg each of bovine serum albumin, ovalbumin, and carbonic anhydrase in a 200-µl volume of T/N Buffer was run under the same condition to provide size standards. An aliqout of each column fraction was analyzed by SDS-PAGE to determine the position of the protein standards.
Samples of TrfA-33
protein alone, of TrfA-N123 alone, and of an approximately 1:1
molar mixture of the two proteins were diluted to a final protein
concentration of 0.125-0.25 µM and incubated on ice
for 30 min and then at room temperature for 10 min. They were then
tested for heterodimer formation by cross-linking with glutaraldehyde
followed by SDS-PAGE and Western analysis. As shown in Fig. 1(panel U), the mixed proteins did not form
heterodimers. Similar results were obtained when the proteins were
incubated on ice overnight (data not shown). However, when the proteins
after mixing were diluted to the same concentration in HEPES Buffer
containing 4 M guanidine HCl, incubated, and then renatured by
dialysis against buffer without guanidine, glutaraldhyde cross-linking
revealed the formation of heterodimers (H; Fig. 1, panel T). This indicated that once a dimer was formed, there
was little dissociation of the subunits but that dimers could reform
after denaturation and renaturation of the protein.
Figure 1:
Glutaraldehyde
cross-linking of TrfA heterodimers formed in vitro. Western
blot using anti-TrfA antiserum of cross-linked TrfA-N123 (N
123), TrfA-33 plus TrfA-N
123 (WT + N
123), and TrfA-33 (WT) proteins that had been
either chemically denatured/renatured in vitro (panel
T) or that were treated but without denaturation (panel
U, untreated control). The positions of TrfA-33 monomer (WT) or homodimer (WT*), TrfA-N
123 monomer (N
123) or homodimer (N
123*), or the
TrfA-33/TrfA-N
123 heterodimer (H) are
indicated.
Figure 2:
Binding of in vitro guanidine-treated TrfA proteins to DNA probes containing one or
two binding sites. Samples of TrfA-33 alone (WT),
TrfA-N123 alone (N
123), or the two proteins mixed
were denatured with 4 M guanidine-HCl then renatured by
dialysis and used in gel mobility shift assays with either a single
binding site probe (lanes 1-3) or a two binding site
probe (lanes 4-15). Treated proteins were added to the
reactions with the single binding site probe as follows: 37.2 ng of
TrfA-33 (lane 1), 50 ng of TrfA-33 + TrfA-N
123 (3:1
molar ratio) (lane 2), and 12.9 ng of TrfA-N
123 (lane
3). For assays with the two binding site probe, increasing amounts
of the proteins were added as follows: TrfA-33 protein at 0.46 ng (lane 4), 0.93 ng (lane 5), 1.86 (lane 6),
and 3.72 ng (lane 7); TrfA-33 + TrfA-N
123 (1:1 molar
ratio) at 0.93 ng (lane 8), 1.86 ng (lane 9), 3.72 ng (lane 10), and 7.44 ng (lane 11) and TrfA-N
123
protein at 0.625 ng (lane 12), 1.25 ng (lane 13), 2.5
ng (lane 14), and 5 ng (lane 15). White
arrowheads indicate the positions of complexes resulting from the
addition of TrfA-33 protein; black arrowheads indicate the
positions of complexes resulting from the addition of TrfA-N
123
protein;
indicates the position of the complex resulting from
the binding of one TrfA-33 and one TrfA-N
123 protein to the probe.
The lower band in all lanes is free probe; the upper band in all lanes is the vector fragment that was also labeled during
probe construction.
When the protein sample
containing a TrfA-33 plus TrfA-N123 mix (which should contain the
TrfA-33 homodimer, the TrfA-N
123 homodimer, and the
TrfA-33/TrfA-N
123 heterodimer) was used with the single binding
site probe, there were only two retarded complexes that migrated to the
same position in the gel as the complexes formed by each protein alone (Fig. 2, lane 2). When a TrfA-33 plus TrfA-N
123
mix was used with the double binding site probe a total of five
complexes were formed (Fig. 2, lanes 8-11). The
two fastest migrating complexes (which should represent binding to one
of the two sites on the probe) ran at the same position as a complex
formed by each protein alone. Of the three slowest migrating complexes
(which resulted from the binding of both sites on the probe), the upper
and lower band corresponded to complexes formed by each protein alone.
The intermediate band was unique to the mixed protein sample. These
results suggest that TrfA binds as a monomer and that the intermediate
band represents the situation where both the truncated and the
wild-type TrfA monomer were bound to the same DNA fragment, which
contains two binding sites.
However, the possibility that TrfA binds
as a dimer and that the heterodimer form is not functional for binding
could not be ruled out from this experiment alone, particularly since
the heterodimers were formed by renaturation of the denatured
homodimers. To test this possibility and to provide additional evidence
for binding by the monomer form, we decided to form heterodimers, in vivo, using a derivative of TrfA-33 that has 36 additional
amino acids at the amino terminus, including a stretch of six histidine
residues. This protein, designated His6-TrfA, is functional in vivo and in vitro for mini-RK2 replication. ()
Using compatible co-resident vectors, either the
His6-TrfA plus TrfA-33 proteins or the His6-TrfA plus TrfA-N123
proteins were expressed in the same cell. Protein was purified from
cleared lysates of these E. coli cultures using a Ni-NTA
affinity column to specifically bind His6-TrfA. This scheme results in
the purification of protein samples that have His6-TrfA homodimers (or
monomers) and His6-TrfA/TrfA-33 or His6-TrfA/TrfA-N
123
heterodimers but no TrfA-33 or TrfA-N
123 homodimers (or monomers).
While these latter homodimers are formed in the cell, they cannot be
purified on Ni-NTA, which requires a stretch of histidine residues for
binding (Fig. 3). Thus, each purified protein sample has only
two dimeric forms of TrfA protein, as compared with the three forms
obtained when the heterodimer was prepared in vitro (Fig. 1).
Figure 3:
Purification of His6-TrfA homodimers and
His6-TrfA/TrfA-33 heterodimers from cells expressing both His6-TrfA and
TrfA-33 proteins. A, Western blot using anti-TrfA antiserum of
160 µg of cleared lysates, separated by SDS-PAGE, prepared from
cultures of E. coli BL21(pBK3), which expresses TrfA-33 (lane 1); BL21(pAT30), which expresses His6-TrfA (lane
2); BL21(pAT35)(pBK3), which expresses both His6TrfA and TrfA-33 (lane 3); and BL21(pBK3) plus BL21(pAT30) cells grown
separately but mixed prior to purification (lane 4). B, proteins purified from the cleared lysates by affinity to a
Ni-NTA resin separated by SDS-PAGE and then stained with Coomassie
Blue: lane 1, BL21(pBK3); lane 2, BL21(pAT30); lane 3, BL21(pAT35)(pBK3); lane 4, BL21(pBK3) plus
BL21(pAT30) cells grown separately but mixed prior to purification; and lane 5, purified His6-TrfA (striped arrowhead) and
TrfA-33 (white arrowhead) proteins. 20 µg of protein was
loaded per lanes 2, 3, and 4. Lane 1 had 2 the amount of column elutate as lane
4.
The affinity-purified protein preparations
were then used in gel mobility shift assays with either a single
binding site or a double binding site probe. For the single binding
site probe, either protein sample (His6-TrfA plus TrfA-N123 or
His6-TrfA plus TrfA-33) gave two bands (Fig. 4, lanes 1 and 5). Since each protein preparation has only two forms
of dimeric protein, His6-TrfA homodimers and His6-TrfA/TrfA-33 (or
His6-TrfA/TrfA-N
123) heterodimers, then either TrfA binds as a
monomer (and some dissociation of the protein dimers and heterodimers
must have occurred) or TrfA binds as a dimer and the heterodimers are
functional for binding. The two complexes present in both lanes 1 and 5 run at the exact same position of the proteins
separately purified (lanes 2-4), which is the expected
result if TrfA binds as a monomer.
Figure 4:
Binding of Ni-NTA purified co-expressed
proteins to DNA probes containing one or two binding sites. Gel
mobility shift assays with the one binding site probe (lanes
1-5; duplicate assays) or the two binding site probe (lanes 6 and 7) were performed using either
co-expressed or previously purified protein samples. Purification of
co-expressed His6-TrfA plus TrfA-N123 proteins results in
His6-TrfA homodimers and His6-TrfA/TrfA-N
123 heterodimers.
Purification of co-expressed His6-TrfA plus TrfA-33 proteins results in
His6-TrfA homodimers and His6-TrfA/TrfA-33 heterodimers (see Fig. 3). Protein was added to each assay as follows: lane
1, 1 µg co-expressed His6-TrfA plus TrfA-N
123 proteins; lane 2, 100 ng of purified TrfA-N
123; lane 3,
100 ng of purified His6-TrfA; lane 4, 100 ng of purified
TrfA-33; lane 5, 1 µg of co-expressed His6-TrfA plus
TrfA-33 proteins; lane 6, 268 ng (a) or 535 ng (b) of co-expressed His6-TrfA plus TrfA-N
123 proteins;
and lane 7, 308 ng (a) or 615 ng (b) of
co-expressed His6-TrfA plus TrfA-33 proteins. Black arrowhead indicates the position of a complex resulting from the binding of
TrfA-N
123 protein; white arrowhead indicates the position
of a complex resulting from the binding of TrfA-33 protein; striped
arrowhead indicates the position of a complex resulting from the
binding of His6-TrfA; and
indicates the position of a complex
resulting from the binding of one His6-TrfA and one TrfA-N
123
protein (lane 6) or one His6-TrfA and one TrfA-33 protein (lane 7) to the two binding site probe. The lower band in all lanes is the free probe; the upper band in all
lanes is the vector fragment, which was also labeled during probe
construction.
With the two binding site probe, there was a maximum of five retarded complexes for either protein preparation (Fig. 4, lanes 6 and 7). These five bands run at the exact same position of the purified proteins when they are mixed together in vitro; four of the bands line up with bands obtained when each of the two proteins were purified separately and then bound to the DNA (data not shown). The only consistent interpretation of these results is that TrfA binds as a monomer.
Figure 5: Activation of His6-TrfA for binding to oriV. Increasing amounts of guanidine-treated/renatured His6-TrfA protein (lanes 8-12) and of the treated but nondenatured control (lanes 2-6) were incubated with 0.4 fmol of oriV probe in the absence of nonspecific competitor DNA. The nondenatured His6-TrfA protein was added at 1.65 ng (lane 2), 5 ng (lane 3), 10 ng (lane 4), 20 ng (lane 5), and 50 ng (lane 6). Denatured/renatured His6-TrfA was added at 0.25 ng (lane 8), 0.5 ng (lane 9), 1 ng (lane 10), 5 ng (lane 11), and 10 ng (lane 12). Lanes 1 and 7 have no protein added.
Figure 6:
Separation of His6-TrfA protein on
Superose-12 sizing column. A, protein profiles of
guanidine-treated/renatured His6-TrfA () and of nondenatured but
treated His6-TrfA (
) run on a Superose-12 column. The relative
amount of His6-TrfA was determined in 100-µl aliquots from each
fraction by an antibody capture assay using anti-TrfA anitserum. The
position of the bovine serum albumin (66 kDa), ovalbumin (44 kDa), and
carbonic anhydrase (29 kDa) protein standards run on the column are
indicated with arrows at the top of the profile. B, gel mobility shift assay using the oriV DNA probe
with 5-µl aliquots of fractions from the
guanidine-treated/renatured His6-TrfA column separation. C,
gel mobility shift assay using the oriV DNA probe with
5-µl aliquots of fractions from the nondenatured His6-TrfA column
separation.
We have previously shown that the replication initiation protein, TrfA, of the broad host range plasmid RK2, binds specifically to the iterons present at oriV(11) . Even though the protein is isolated largely as a dimer(11, 18, 19) , in this paper we show that TrfA binds to iteron DNA as a monomer (Fig. 2, Fig. 4, and Fig. 6). Moreover, TrfA dimers are very stable ( Fig. 1and Fig. 3), at least in the various buffers used, with no detectable exchange of monomers when different sized TrfA molecules, either purified or in crude cell lysates, are mixed. Formation of heterodimers of different sized TrfA proteins requires either in vitro renaturation of chemically denatured proteins (Fig. 1) or co-expression of the different sized proteins in vivo (Fig. 3).
The cI-TrfA fusion
protein constructed in this study was used in a biological assay for
repressor activity to demonstrate that TrfA can form dimers in vivo. However, since the construct expressing the cI-TrfA
fusion protein expressed significantly more protein then native RK2
(data not shown), it is not clear whether or not the TrfA protein is
dimeric in E. coli cells carrying intact RK2. An analysis of
exponentially growing E. coli cells carrying RK2 determined
that there are 220 monomeric of TrfA-33 and 80 molecules of TrfA-44 per
cell(36) . Assuming a cell volume for exponentially growing E. coli cells of 4.6
10
µl(41) , the in vivo concentration of TrfA
is 1.08 µM. The results presented in Fig. 5show
that protein at 0.27 µM could be activated for binding
approximately 40-fold; this activation is presumably the result of the
conversion of dimers to monomers. Also, as shown in Fig. 6,
His6-TrfA at 1.35 µM was primarily dimeric even after
treatment to enhance monomer formation. Thus, assuming there is no
active process in the cells that converts dimers to the monomer form,
it seems likely on the basis of the in vivo estimates of TrfA
concentration and the in vitro observations presented here
that unbound TrfA is largely present as a dimer in E. coli.
Attempts to utilize the cI-TrfA fusion protein system to isolate
TrfA dimerization-defective mutants by replacing the wild type trfA sequence in pSP32 with previously mutagenized trfA sequences (31) have not been successful. ()The
use of this dimerization reporter system for this type of screen was
described in the original report of the system(40) . Subsequent
studies have used cI reporter systems to isolate or characterize
cellular proteins that by their binding to the cI-fusion protein
interfere with the dimerization of yeast GCN4(40) , HIV
Tat(42) , or Myc(43, 44) . With the TrfA
dimerization mutant screen, the few E. coli(pSP28-mutant)
clones initially isolated that expressed full-length cI-TrfA fusion
protein and were consistently defective in dimerization as determined
by their sensitivity to
infection proved upon DNA sequencing to
have large regions of DNA sequence rearrangements in trfA. The
same bank containing the mutagenized trfA genes has been
utilized successfully in other mutant
screens(18, 31, 45, 46, 47) ,
where all mutants isolated were found to result from single point
mutations. While more can be done with this approach, the results to
date suggest that a single point mutation is not likely to abolish the
ability of the TrfA protein to dimerize.
Functional roles have been shown for both the dimer and monomer forms of the initiation proteins of plasmids F and pSC101, with dimers binding to indirect repeats at the rep promoter and monomers binding to the origin iterons(26, 27) . Evidence has also been obtained for pSC101 indicating that dimeric RepA bound to the inverted repeat sequence, IR1, interacts with monomeric RepA bound to the iterons, and this interaction is an element in the control of plasmid copy number (48) . For plasmid P1, a role has not yet been identified for the dimer form of the RepA protein, and results have been obtained that suggest that in vivo RepA is primarily monomeric(49) .
The functional significance of the dimer form of TrfA is unclear.
Expression of trfA is not subject to autoregulation, and no
indirect repeats related to the iteron sequences are present upstream
of trfA. Two possible half-sites related to the RK2 iterons
have been identified elsewhere in RK2 (15) however, there is no
evidence for the binding of TrfA protein to these half sites. ()
TrfA protein has been shown to have both a positive and
a negative activity in regulating replication from the RK2
origin(31, 50) . By binding to the origin iterons,
TrfA activates replication. The kinetics of TrfA dimer dissociation
into monomers might influence the rate of assembling of the replication
initiation complex on oriV, thus adding an additional level of
control for replication initiation. However, the copy number of plasmid
RK2 is not increased in response to increases in TrfA protein
levels(36) . Alternatively or additionally, TrfA dimerization
may be involved in the regulatory function of TrfA in plasmid copy
number control. It has been proposed that, when the concentration of
RK2 iteron-containing origins in the cell exceeds the typical plasmid
copy number, all of the TrfAoriV complexes are
reversibly coupled at their origins thereby preventing DNA replication
(handcuffing model)(31, 36, 50) . It is
possible that the dimeric form of TrfA plays a role in the reversible
coupling of the monomeric TrfA
oriV complexes.
The E. coli chaperon proteins DnaJ and DnaK are apparently
involved in the formation of monomers from dimers of the RepA
initiation protein of P1 (28) and the RepE initiation protein
of plasmid F(27) , although an argument has been made for P1
RepA that the role of the chaperons is to activate inactive
monomers(49) . The DnaK protein may also be important for
pSC101 replication(26) . Unlike the case with these other
initiator proteins, incubation of His6-TrfA or TrfA-33 with the E.
coli DnaJ and DnaK proteins in vitro has no affect on the
activity of the TrfA protein in binding to the iterons at the origin of
replication. ()In addition, studies with E. coli DnaJ and DnaK deletion mutants indicate that these two chaperones
are not required for RK2 replication in vivo. (
)It
is possible, however, that other E. coli chaperone proteins,
such as GroEL, are involved in TrfA activation. Alternatively, the
conversion of TrfA dimers to monomers in vivo may simply be
passive. Given the stability of TrfA dimers in solution, it is possible
that it is not the dissociation of dimeric TrfA that provides the
source of monomeric TrfA available for DNA binding, rather it is de
novo synthesized TrfA that binds DNA prior to dimerization. The
isolation of a dimerization defective mutant of TrfA would, therefore,
be important for determining the exact mechanism of dimer to monomer
conversion and certainly would be very useful in determining the role
of TrfA dimers in RK2 replication.