From the Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D. C. 20057
Received for publication, September 12, 2002, and in revised form, October 28, 2002
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ABSTRACT |
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Dominant negative forms of the phage Mu
repressor, including the mutant Vir repressors, are not only rapidly
degraded by the ClpXP protease but also promote degradation of the
unmodified, wild-type repressor. This trans-targeting of
the wild-type repressor depends upon a determinant within its
C-terminal domain, which is needed for recognition by ClpX. An
environmentally sensitive fluorescent probe
(2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS)) attached
to the C terminus of the full-length repressor indicated that Vir
induces the movement of this domain into a more exposed configuration.
Vir also promoted attachment of MIANS to the C terminus of the
repressor at an accelerated rate, and it greatly increased the rate of
phosphorylation of a cAMP-dependent protein kinase motif
attached to the repressor C terminus. While an excess of Vir was needed
to promote repressor phosphorylation at maximal rates, the presence of
ClpX could increase phosphorylation rates at lower Vir levels.
trans-Targeting of the Mu repressor is therefore promoted
by exposing its ClpX recognition determinant, and the action of ClpX
can assist Vir in exposing these determinants.
Degradation of substrates by the Escherichia coli ClpXP
protease is carried out by recognition of the substrates by the
molecular chaperone ClpX, which promotes the ATP-dependent
unfolding and transfer of the substrates to the active site of the ClpP
subunit (1-4). The proteolytic component is made up of a tetradecamer of ClpP subunits, which form an inner proteolytic chamber where the
serine active sites are located (5). ClpX, which forms a hexameric ring
(6), binds proteins that bear a recognition motif found either at the N
or C terminus of natural substrates (7-9), a determinant for which no
apparent consensus sequence has yet emerged.
One mechanism for regulating degradation of ClpXP substrates is
trans-targeting, in which a regulatory protein promotes
degradation of a second protein. Substrates of this targeting mechanism
include the DNA polymerase subunit UmuD', the stationary phase and
stress response sigma factor
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S, peptides marked with the
ssrA degradation tag, and the phage Mu immunity repressor
(Fig. 1A). Proteolysis of
these proteins is promoted respectively by the UmuD' precursor UmuD
(10), RssB (11-14), SspB (15), and dominant negative forms of phage Mu
repressor called Vir1
(16-18). The mutant Vir repressors have an altered sequence at the C
terminus due to a frameshift mutation (Fig. 1C). They not only are rapidly degraded by ClpXP but also promote degradation of the
unmodified repressor (hereby called Rep to indicate the wild-type
repressor). Repressor peptides marked with the ssrA tag can
also promote degradation of Rep (19).
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Fig. 1.
Repressor domains and mutants.
A, the three major domains of the repressor. The
leucine-rich domain located between the DBD and the CTD is hypothesized
to play a role in repressor oligomerization (38) and is required for
repressor activity (19, 47, 48). B, repressor proteins with
a single cysteine at the C terminus for the attachment of MIANS. There
is a C17A change in the DBD so that MIANS does not bind at that site.
C, sequence at the CTD of wild-type, mutant, and engineered
repressors. Those residues that are different from the Rep sequence are
marked with and an asterisk.
For the Mu life cycle, trans-targeting can potentially be a mechanism for promoting derepression in response to specific physiological conditions. The Mu genome is a transposable element, a property that is exploited in the establishment of lysogeny and phage DNA replication (see Ref. 20 for a review). Rep, encoded by the c gene, establishes lysogeny by shutting down Mu transposition functions A and B after the first integration event. Binding to Mu operator sequences, Rep shuts down transcription of the transposition genes (21) and also competes for MuA transposase binding sites within the operator (22, 23). Degradation of the Mu repressor induced by co-expression of Vir can trigger lytic development. Physiological conditions that promote derepression include carbon starvation and entry into stationary phase, a process termed S derepression, which is dependent upon clpX, clpP, lon, and rpoS host functions (24). Although Mu lytic development does not proceed at optimal rates under these conditions, derepression can lead to transposase-dependent genetic rearrangements (25-27). An interesting possibility is that ssrA-tagged repressor peptides, which can arise when ribosomes become stalled on mRNA (28-30), may trigger repressor degradation under starvation and stationary phase conditions (19).
Rep is stable in vivo even though it is degraded by ClpXP protease with approximately the same catalytic rate constant as that of Vir (31). Unlike Vir, Rep is degraded with a relatively high Michaelis constant (Km), and it becomes resistant to ClpXP in the presence of DNA. In the presence of Vir, Rep acquires the attributes of low Km and protease sensitivity in the presence of DNA. These results have suggested that a determinant recognized with high affinity by ClpX is already present in Rep but is not readily accessible, especially in the presence of DNA. trans-Targeting proteins such as Vir may function to expose such a determinant in Rep to promote its degradation. The self-associating property of repressor molecules, which exists in solution as a hexamer or higher order oligomer (32, 33), may promote Rep-Vir interactions for this process.
We have previously hypothesized (31, 34) that such interactions may
induce movement of the repressor C-terminal domain (CTD, residues
Ile-170 to Val-196; see Fig. 1A), which functions in
modulating Rep degradation as well as DNA binding. Deletion of 18 residues from the C terminus (Rep18 encoded by the
sts62-1 allele; Fig. 1C) suppresses
temperature-sensitive mutations in the N-terminal DNA binding domain
(DBD; Lys-13 to Ser-80) and confers dominance over the vir
alleles (35). In ssrA knock-out strains, truncated
repressors like Rep
18 accumulate and block S derepression (36).
There are a number of lines of evidence that indicates that the CTD
includes key residues that make up the ClpX recognition determinant.
Repressor mutants with an 18-residue deletion (Rep
18) or certain
single amino acid replacements in the CTD have been found to be
resistant to ClpXP degradation in the presence or absence of Vir (37).
In addition, heat denaturation accelerates Rep degradation by ClpXP
without Vir, while ClpXP-resistant CTD mutants cannot be degraded even
after denaturation.
In this paper we demonstrate that Vir promotes conformational changes
in Rep, inducing movement of the CTD such that it becomes more exposed.
We previously determined that the Rep C terminus is found in a
relatively hydrophobic environment, whereas the Vir C terminus is found
in a hydrophilic environment (34). Here we demonstrate that Vir can
induce the Rep C terminus to enter an even more hydrophilic
environment, allowing enzymes to gain access to this domain more
readily. Moreover, the action of ClpX is able to reduce the number of
Vir molecules needed to promote maximal accessibility. The results
indicate that Vir promotes trans-targeting by exposing ClpX
recognition determinants within the CTD and that ClpX can assist
Vir in this process.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains, Plasmids, and Proteins--
Repressor
proteins Rep and Vir3060 were overexpressed from plasmids pHS502 and
pHS504, which contain the respective repressor alleles between the
NdeI and BamHI sites of pET-11a (Novagen). Plasmids pGTN130 and pGTN206 for overproducing RepC17A and VirC17A were
constructed from pHS502 and pHS504, respectively, using the Stratagene QuikChangeTM site-directed mutagenesis kit.
Construction of plasmids pGTN204 (RepC197) and pGTN211 (VirC192),
derivatives of pREP-1 (31), and pGTN210 (Rep18), a derivative of
pHS502, were described previously (34). RepNK was overproduced from
pREP-NK, which was constructed by PCR amplification of the
c+ gene in pJV300 (38), using an N-terminal
primer that contains the coding sequence for the
cAMP-dependent kinase motif (39). The primer adds the
sequence GGACTTCGAAGAGCTTCTGTTATC encoding the peptides GIRRASVI
between the first two codons (Met1 and Lys2) of
the repressor gene. The RepNK coding sequence was inserted at the
NcoI and BamHI sites of pET-19b (Novagen), using
the NcoI site at the AUG start codon and a BamHI
site adjacent the stop codon. RepCK was overproduced from pREP-CK,
which is a derivative of pREP-1. The sequence CTTCGAAGAGCTTCTGTTGGTUAA
encoding the peptide IRRASVG was fused to the C-terminal coding
sequence of the c+ gene using the Stratagene
ExSiteTM PCR-based mutagenesis kit.
All repressor proteins (34) and the ClpX protein (31) were purified as described previously. All protein concentrations are given in equivalents of protein monomers.
Fluorescence Spectroscopy-- Thiol-specific attachment of 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS; Molecular Probes) to repressor proteins and quantitation of bound MIANS were conducted as described previously (34). A 3-6-fold excess of MIANS over the target repressor protein was added to each reaction mixture, which was incubated at 37 °C. In all experiments, greater than 90% of the target repressor proteins, which bear a single cysteine residue, had bound MIANS after incubation for 10 min.
Fluorescence measurements were taken with an Aminco-Bowman Series 2 luminescence spectrometer using a path length of 4 cm. All analyses were conducted in 25 mM HEPES-K+, 200 mM potassium glutamate, and 10 mM maganesium acetate with the indicated concentrations of repressor proteins in a sample volume of 200 µl. MIANS fluorescence was monitored at an excitation wavelength of 330 nm and an emission wavelength of 420 nm with constant stirring at 37 °C.
Phosphorylation of RepNK and RepCK--
RepNK radiolabeled to
high specific activity for trypsin cleavage analysis was prepared as
described previously (39) with modifications. Reaction mixtures (30 µl) contained 3 µM RepNK, 1.5 µM
[-32P]ATP (PerkinElmer Life Sciences; 3000 Ci/mmol), 25 mM Tris-HCl (pH 7.5), 2 mM DTT,
100 mM NaCl, 12 mM MgCl2, 10 mM NaF, and 25 units catalytic subunit of the
cAMP-dependent protein kinase (PKA; New England Biolabs).
Incubation was at 37 °C for 10 min, and the reaction was stopped by
addition of EDTA to 30 mM.
The kinetics of phosphorylation of RepNK and RepCK was monitored under
the same conditions except that the reaction mixture contained 60 µM [-32P]ATP (10 Ci/mmol), 8 units of
PKA, and repressor proteins at the indicated concentrations. When Vir,
Rep, or ClpX were present together with RepNK or RepCK, the reaction
mixture was assembled and allowed to incubate 10 min at 37 °C prior
to adding [
-32P]ATP and PKA. Reaction mixtures were
resolved by electrophoresis on a 10% or 12% SDS-polyacrylamide gel.
The gel was stained with Coomassie Blue R-250 and dried down for
quantitation on an Amersham Biosciences Storm 840 PhosphorImager
system. Bands corresponding to the maximum incubation times were then
excised, and the level of Cheronkov radiation was measured on a Beckman
LS6500 Multi-purpose Scintillation Counter to determine the fraction of
repressor proteins phosphorylated.
Trypsin Cleavage of Repressor Proteins--
Reaction mixtures
(20 µl) contained 25 mM HEPES-K+ (pH 7.5),
200 mM potassium glutamate, 10 mM magnesium
acetate and the indicated concentrations of
32P-RepNK (10,000-13,000 cpm/pmol) and unlabeled
repressor proteins. 32P-RepNK and an unlabeled
repressor protein were first mixed and allowed to incubate for 10 mins
at 37 °C before addition of 1 µg trypsin. Digestion was allowed to
proceed at the same temperature for 1 h, and then reactions were
stopped by the addition of phenylmethylsulfonyl fluoride to 2% (w/v)
final concentration. The cleavage patterns were analyzed on a 10%
SDS-polyacrylamide gel. The gel was stained with Coomassie Blue,
dried down, and subjected to autoradiography.
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RESULTS |
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Mutant Repressors with an Altered CTD Induce Conformational Changes
in Rep--
We examined whether mutant repressors with an altered CTD
can influence the conformation of Rep molecules. We first examined their influence on the conformational state of Rep using the trypsin hypersensitive site at Arg-70 as a marker (34). When not bound to DNA,
Rep is hypersensitive to cleavage at Arg-70, a property dependent upon
the close proximity of the CTD to the DBD. Upon cleavage at Arg-70, the
resulting peptides become susceptible to cleavage by trypsin at other
sites. This hypersensitivity, which is lost as Rep interacts with DNA
and the CTD moves away from the DBD, is absent in Vir and Rep18.
These repressor molecules therefore remain intact under conditions that
promote degradation of Rep to small fragments.
We determined that both Vir and Rep18 could induce Rep to lose
hypersensitivity at this site. Purified wild-type repressor with a
cAMP-dependent protein kinase motif at the N terminus
(RepNK) was radiolabeled using [
-32P]ATP, mixed with
an excess of unlabeled Rep, Vir, Rep
18, or RepNK, and treated
with trypsin. The susceptibility of 32P-RepNK to
tryptic digestion was influenced by the properties of the excess
unlabeled repressor protein in the reaction mixture. 32P-RepNK in the presence of unlabeled Rep and RepNK
was rapidly cleaved by trypsin (Fig. 2,
lanes 5 and 8). In contrast, Vir and Rep
18
remained relatively resistant to cleavage and
32P-RepNK became resistant in their presence
(lanes 6 and 7). This indicates that Vir and
Rep
18 can induce conformational changes in Rep, most likely by
inducing movement of the CTD with respect to the DBD.
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Vir Induces Movement of the Rep CTD to a More Hydrophilic
Environment--
We previously determined that the Rep C terminus is
in a hydrophobic environment, whereas the Vir3060 C terminus is in a
relatively hydrophilic one (34). We used repressor proteins with the
C17A substitution and a single cysteine introduced at the C terminus, modifications that do not alter the respective properties of the protein as a ClpXP substrate (data not shown; VirC192 and RepC197; see
Fig. 1B). As demonstrated previously, when the
thiol-specific fluorophore MIANS was attached to the single cysteine at
the C terminus, we obtained emission maxima of 429 and 439 nm for
RepC197 and VirC192, respectively (Fig.
3, A and B). The
fluorescence intensity of VirC192-MIANS was greatly diminished in
comparison with that of RepC197-MIANS, consistent with the probe at the
Vir C terminus being in a more hydrophilic environment (40).
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When MIANS was attached to RepC197 in the presence of 3-fold excess VirC17A, which has no cysteine residues, fluorescence intensity was diminished (Fig. 3A), and the emission spectrum was red-shifted 20 nm to 449 nm (Fig. 3B), indicating that the Rep C terminus had entered an environment even more hydrophilic than that of the Vir C terminus. Although RepC197-MIANS in the presence of Vir has an emission maximum of longer wavelength, it has a higher fluorescence intensity than VirC192-MIANS. At the C terminus of Vir, MIANS is attached to basic residues (HRR), and these residues most likely further quench its fluorescence. The presence of 3-fold excess RepC17A instead of VirC17A slightly increased the fluorescence intensity and did not change emission maximum of the attached MIANS (Fig. 3C). The results indicate that Vir can induce movement of the Rep CTD such that it is more exposed to solvent and therefore more accessible to ClpX. Reducing the levels of VirC17A to a level equimolar to RepC197 diminished the red shift to ~1-2 nm (data not shown). An excess of Vir was therefore needed to efficiently induce the Rep conformational change.
Vir Increases the Rate of MIANS Attachment to the Rep C
Terminus--
MIANS has the convenient characteristic of becoming
fluorescent only upon attachment to cysteine residues, and we used this property to examine accessibility of Rep C terminus. MIANS is attached
to VirC192 at a much faster rate than it is to RepC197 (34). In the
presence of VirC17A, the rate of MIANS attachment to RepC197 was
increased to the same rate as its attachment to VirC192 (Fig.
4A), indicating that the
Vir-induced movement of the Rep CTD greatly increases its
accessibility. In the attachment of MIANS to VirC192, a spike in
fluorescence was reproducibly recorded at ~200 s (34), an anomaly not
observed in the attachment of MIANS to RepC197 when VirC17A was present
(Fig. 4A). Although the cause of this transient increase in
fluorescence has not been definitively demonstrated, it may reflect a
transient fluctuation in the conformation of the VirC192 protein
population upon becoming saturated with MIANS.
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Unlike its effect on RepC197, VirC17A decreased the rate of attachment of MIANS to the native C17 residue present in the DBD of Rep (Fig. 4A). This indicates that VirC17A is not increasing accessibility of all parts of Rep. Moreover, RepC17A had no effect on the attachment of MIANS to RepC197 (Fig. 4B).
ClpX Decreases the Amount of Vir Required to Promote Maximal
Accessibility of the Rep C Terminus--
The 7-residue
cAMP-dependent protein kinase motif (IRRASVG) was
engineered onto the C terminus of Rep (RepCK) to examine how Vir and
ClpX can influence the rate of its phosphorylation. Under the assay
conditions, RepCK was phosphorylated at a very low rate, and the
presence of ClpX did not significantly increase the rate of
phosphorylation (Fig. 5A).
However, Vir promoted a large increase in the rate of RepCK
phosphorylation (Fig. 5A). Increasing the Vir to RepCK ratio
from 1:1 to 4:1 progressively increased the rate of phosphorylation
(Fig. 5, A-D), indicating that the Rep CTD movement induced
by Vir exposes this domain and makes the attached kinase motif
accessible to phosphorylation. At a Vir to RepCK ratio of 4:1, the
presence of ClpX promoted only a slight increase in the phosphorylation
rate; but at lower levels of Vir, ClpX did promote a significant
increase in phosphorylation rates, indicating that the recognition and
unfolding of protomers in repressor oligomers by ClpX can further
expose the CTDs of additional Rep molecules. In contrast, Vir could not
promote any increase in the low level of phosphorylation of the kinase
motif at the Rep N terminus even in the presence of ClpX (Fig.
5E), indicating that Vir and ClpX are not necessarily
increasing accessibility of all domains of Rep. In addition, Rep and
Rep18 were unable to promote any increase in the phosphorylation
rate of RepCK (Fig. 5F), indicating that these proteins do
not promote exposure of the Rep CTD. These results indicate that ClpX
can assist Vir in exposing the Rep CTD.
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DISCUSSION |
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trans-Targeting of Rep by Vir Is Promoted by Inducing Conformational Changes That Expose ClpX Recognition Determinants in the Rep CTD-- The CTD of Rep plays a critical role in both Rep degradation promoted by altered forms of the repressor and the formation of the repressor-operator complex, enabling the Rep population to respond to specific physiological conditions such as carbon starvation and entry into stationary phase (19, 24, 35, 36). Although ClpXP-sensitive Vir can promote Rep degradation, Rep does have intrinsic sensitivity to ClpXP protease by itself (31), indicating that it contains a ClpX recognition determinant. However, the context of this determinant influences to a large degree the efficiency with which it is recognized by ClpX. In the absence of DNA, Rep is degraded with a relatively high Km. In the presence of Vir, Rep is degraded with a lower Km that is essentially identical to that of Vir. Heat denaturation of Rep also allows it to be rapidly degraded without Vir (37).
Thus, what constitutes a ClpX recognition determinant is not simply
determined by the presence of a recognition motif but also by factors
such as accessibility and display of the motif. And what constitutes
the minimal elements of the Rep ClpX recognition determinant remains to
be defined. Nevertheless, the Rep CTD is an important part of that
determinant. Cysteine replacement mutations at 8 of the last 17 C-terminal residues and a deletion of even a single residue at the C
terminus result in gain of dominance over vir alleles (37).
Biochemical analyses have shown that Rep18, and at least two CTD
mutants with a single cysteine replacement (V183C and V196C) have lost
intrinsic sensitivity to ClpXP even after heat denaturation, indicating
that a determinant within the CTD is required for ClpX recognition. Vir
can induce the movement of the Rep CTD such that it is more exposed,
most likely so that it can be bound by ClpX. Thus
trans-targeting can be the result of unmasking a largely
inaccessible recognition determinant.
Although an excess of Vir was needed to induce the conformational change, ClpX could assist in the Vir-induced exposure of the CTD. Very low levels of Vir must be effective in promoting derepression, for it is rapidly degraded in vivo and is not able to accumulate to high levels and yet it effectively promotes Rep degradation (17). Vir promotes recognition of Rep molecules by ClpX, which in turn aids Vir in inducing Rep conformational changes. Recognition and unfolding of protomers may dissociate ClpXP-sensitive repressor oligomers and create interacting surfaces for conversion of additional Rep molecules to the protease-sensitive conformation. The repressor molecules that induce the conversion to the sensitive conformation may not necessarily be Vir molecules and may be Rep molecules previously converted to this conformation.
If Vir and ClpX catalytically generate Rep molecules with an exposed CTD, it is likely that such Rep molecules maintain this "exposed" conformation only transiently before returning to the "unexposed" conformation. For example, at a limiting Vir concentration, ClpX promotes increased phosphorylation of RepCK at a uniform rate over a 25-min time course (Fig. 5D). If stably exposed RepCK molecules accumulate during the time course, phosphorylation of RepCK should be accelerated in this time period. The presence of Vir and ClpX therefore appears to increase phosphorylation rates by maintaining a higher steady-state level of exposed RepCK. Less than stoichiometric amounts of Vir that efficiently promote Rep degradation in vitro (31) are not as effective in increasing RepCK phosphorylation. However, if the recognition and unfolding of Rep protomers lead to exposure of additional ClpX recognition motifs, protein unfolding by ClpX may be coupled to its recognition of additional protomers. In this way, when a threshold of inducing molecules such as ssrA-tagged repressor peptides accumulate under certain physiological conditions, they may begin a catalytic process to promote degradation of the bulk Rep population.
Implications for Understanding General Mechanisms Involved in trans-Targeting-- It is not yet clear whether the mechanism of trans-targeting of the Mu repressor by Vir will turn out to be similar to trans-targeting by other regulator proteins that promote degradation. Unlike Vir, trans-targeting proteins UmuD, RssB, and SspB target the respective substrates without being degraded themselves by ClpXP (10, 14, 15). Gonzalez et al. (10) determined that an alanine stretch introduced into the UmuD N-terminal segment, which is not present on UmuD', prevents UmuD-directed degradation of UmuD' by ClpXP. The result suggests that this is the segment recognized by ClpX in the UmuD-UmuD' heterodimer. UmuD as well as RssB and SspB may interact with ClpX to deliver the respective protease substrates to the active site of ClpX.
Nevertheless, ClpX substrates in general have been found to contain
recognition motifs, for which there is no apparent consensus sequence.
Thus, it is conceivable that these regulator proteins promote exposure
of a motif within the target protein. For example, S and
phosphorylated RssB together bind with high affinity to ClpX even
though neither alone do so (14); this leaves open the possibility that
the complex exposes a determinant in
S bound by ClpX.
Hoskins et al. (41) recently determined that the ClpX
recognition motif present at the C terminus of MuA transposase can also
be recognized at an interior position of a MuA-green fluorescent
protein fusion, albeit less efficiently. This raises the possibility
that a trans-targeting protein might promote exposure of a
recognition motif present in the interior of the target protein.
Transmission of Conformation Changes as a General Mechanism in
Disseminating Physiological Signals--
Studies of prions in mammals
and prion-like proteins in yeast have raised interesting questions
about the dissemination of conformational states among protein
molecules and whether such a mechanism is generally employed in nature
(42-46). While prions have been implicated in the transmission of a
neurodegenerative disasease (spongiform encelopathy) in mammals,
prion-like proteins in yeast have been implicated in the dissemination
of heritable phenotypic traits. Although Vir is not known to promote
protein-based inheritance, the way it promotes Rep degradation by
inducing conformational changes reflects a prion-like mechanism at work
in the dissemination of a molecular signal.
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ACKNOWLEDGEMENTS |
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We thank Shawna Lagan and Charan Gowda for constructing overproducers of repressor proteins with an attached kinase motif and also C. Gowda for purifying these proteins. We thank Elliott Crooke and Stella North for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM58265 (to H. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, School of Medicine, Georgetown University, Box
571455, Washington, D. C. 20057-1421. Tel.: 202-687-1442; Fax:
202-687-7186; E-mail: nakai@bc.georgetown.edu.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M209352200
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ABBREVIATIONS |
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The abbreviations used are:
Vir, repressor encoded by a virulent mutant of Mu;
VirC192, Vir repressor
with a C17A alteration and a cysteine added at the C terminus;
VirC17A, cysteine-less Vir repressor having a C17A alteration;
CTD, C-terminal
domain;
DBD, DNA binding domain;
MIANS, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid;
Rep, wild-type Mu
repressor;
Rep18, repressor truncated by 18 residues at the
C-terminal end;
RepC197, repressor with a C17A alteration and a
cysteine residue added at the C terminus;
RepC17A, cysteine-less
repressor having a C17A alteration;
RepNK, repressor with a kinase
motif at the N terminus;
RepCK, repressor with a kinase motif at the C
terminus;
PKA, catalytic subunit of the cAMP-dependent
protein kinase.
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