From the Department of Biochemistry and the
M.D./Ph.D. Program, University of Wisconsin-Madison,
Madison, Wisconsin 53706
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ABSTRACT |
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Tn5 is unique among prokaryotic
transposable elements in that it encodes a special inhibitor protein
identical to the Tn5 transposase except lacking a short
NH2-terminal DNA binding sequence. This protein regulates
transposition through nonproductive protein-protein interactions with
transposase. We have studied the mechanism of Tn5
inhibition in vitro and find that a heterodimeric complex between the inhibitor and transposase is critical for inhibition, probably via a DNA-bound form of transposase. Two dimerization domains
are known in the inhibitor/transposase shared sequence, and we show
that the COOH-terminal domain is necessary for inhibition, correlating
with the ability of the inhibitor protein to homodimerize via this
domain. This regulatory complex may provide clues to the structures of
functional synaptic complexes. Additionally, we find that
NH2- and COOH-terminal regions of transposase or inhibitor
are in functional contact. The NH2 terminus appears to
occlude transposase homodimerization (hypothetically mediated by the
COOH terminus), an effect that might contribute to productive transposition. Conversely, a deletion of the COOH terminus uncovers a
secondary DNA binding region in the inhibitor protein which is probably
located near the NH2 terminus.
Transposable DNA elements occur widely in nature and have
important roles in evolution, chromosome mutagensis, and gene transfer. Common to all transposable elements is a need to fix transposition at
low levels to avoid excessive mutagenesis of the host genome.
Transposition is a multistep process involving nucleoprotein
intermediates which is catalyzed by an element-encoded transposase (Tnp).1 The first step is the
specific binding of Tnp to short transposon DNA end sequences that are
inverted repeats. Synapsis follows such that Tnp-Tnp interactions
bridge the ends. Subsequently, the DNA cutting and joining reactions of
transposition occur (for a review, see Ref. 1). In the case of
transposon Tn5, Tnp completely releases the element from the
donor DNA molecule before inserting it into a target DNA site (2).
Tn5 is a 5.8-kilobase composite transposon found in
Gram-negative bacteria which consists of two insertion sequences,
IS50R and IS50L (for reviews, see Refs. 3 and 4).
The insertion sequences, 1.5 kilobases in length, are very nearly
identical and are arranged in inverted orientation flanking a central
region containing antibiotic resistance genes. The outermost ends of the Tn5 transposon overall are 19-bp inverted repeats called
outside ends (OE). IS50R encodes a 53-kDa Tnp protein, 476 amino acids in length, and a 48-kDa inhibitor protein (Inh), 421 amino
acids in length.
Inh is encoded in the same reading frame as Tnp but is transcribed from
a separate promoter and is translated from a distinct initiation codon
such that Inh lacks the first 55 amino acids of Tnp. Inh, apparently,
does not bind to OE DNA, as does Tnp (5), but it can participate in a
three-molecule complex with an OE-bound Tnp monomer (5, 6). Whereas Tnp
acts in cis (i.e. acts on local DNA sequences
relative to its own gene), Inh is a trans-acting factor that
functions post-translationally to form nonproductive complexes with Tnp
(5). Tnp has also been shown to function as a self-inhibitor in
trans (7, 8). Tnp and Inh are folded similarly in terms of
gross secondary and tertiary structure (9), and thus the convention for
numbering the primary amino acid sequence of Inh is based on the Tnp
primary sequence (i.e. the initiating methionine of Inh is
referred to as residue 56). Tnp and Inh share two distinct dimerization
domains2 (9, 10), one of
which is located at the COOH terminus and the other near amino acids
114-314.
Correlating with the fact that Inh-DNA binding has not been detected,
the NH2 terminus of Tnp has been implicated in OE DNA recognition (9-11). A series of NH2-terminal point
mutations at residues 41, 47, and 54 in Tnp has been shown to enhance
OE DNA binding affinity or specificity (11). Also, OE DNA can protect the first 113 amino acids of Tnp against proteolysis (9). Furthermore, the deletion of a very short part of the NH2 terminus
disrupts Tnp-DNA binding (10). Additionally, a predicted
helix-turn-helix DNA binding motif is present in the
NH2-terminal region at residues 35-54.3
Tnp regions constituting a conserved catalytic domain have been
predicted and verified within proteolysis-resistant segments spanning
amino acids 93-217 (9) and 312-365 (12).2 Part of the
catalytic domain, therefore, overlaps with the more NH2-terminal of the two known dimerization regions.
In vitro, Tnp is the only protein necessary for the
catalysis of transposition in the presence of divalent metal ions
(2).
Many transposition systems are regulated negatively by repression of
transcription, regulation of translation, innate Tnp instability or
inactivity, or by accessory protein competition for DNA binding;
however, transposase inhibition by nonproductive multimerization may be
unique to Tn5, especially in prokaryotic systems. Here, the
molecular mechanism of Tn5 inhibition is explored. We
demonstrate a critical importance of the COOH terminus of Tnp/Inh for
inhibition in vitro. Because the two proteins are nearly
identical, the structure of the inactive complex bound to DNA could
provide clues to the nature of functional transposition complexes. An alternative hypothesis separates the inhibitory and synaptic functions of the Tnp protein into distinct regions, inhibition at the COOH terminus and synapsis at an unknown location. Furthermore, we find that
the NH2- and COOH-terminal regions of either Tnp or Inh are
in close contact or proximity, and we discuss possible functional
consequences of this structural arrangement.
Purification of Inh, Tnp, and TnpEK54/LP372--
The
overexpression and purification of Inh and Tnp in Escherichia
coli have been described previously (9). TnpEK54/LP372 was
purified similarly except Heparin HyperD (BioSepra) was used during the
heparin chromatography step. Based on Coomassie-stained SDS-polyacrylamide gels, Inh was >95% pure, Tnp was >90% pure, and
TnpEK54/LP372 was >95% pure. The proteins were stored in 0.4 M NaCl TEG (20 mM Tris-HCl, pH 7.9, 1 mM EDTA, 10% glycerol) at Purification of His6 Fusion Proteins--
Fusion
protein vectors encoding a 41-amino acid leader sequence with a
His6 tag and a protein kinase recognition site were constructed for Inh, InhEQ451, InhAD466, Inh In Vitro Inhibition--
The in vitro assay for
Tn5 transposition has been described (2); however, we used
modified OE with mutations at positions 10, 11, 12, and 15 which are
hyperactive (14) in conjunction with a modified DNA substrate useful
for the detection of cleavage events. This 4-kilobase DNA substrate,
pUC19 (15)-based pGRST2, contains 1.3 kilobases of "transposon" DNA
consisting of the kanamycin resistance gene from Tn903
flanked by the modified OE 19-bp ends. A unique XhoI
restriction site is located 180 bp from one transposon end within the
transposon. The functional version of Tnp used in this assay has a
double mutation, E54K/L372P, which renders a hyperactive,
trans-active transposition phenotype. Transposition reactions contained 0.1 M potassium glutamate, 25 mM Tris acetate, pH 7.5, 10 mM
Mg2+, 50 µg/ml bovine serum albumin, 0.5 mM
Gel Filtration Analysis of Inh and Tnp--
The potential for
homodimerization of Tnp or Inh was assayed by gel filtration. In the
presence of 10% glycerol, Inh was tested in 0.1 M and 0.4 M NaCl, whereas Tnp was tested in 0.4 M NaCl only because of its tendency to precipitate at lower salt
concentrations. For these studies, a Toyopearl TSK HW 55 superfine
(Supelco) 200-ml column was equilibrated with 0.1 M NaCl or
0.4 M NaCl TEG and was calibrated with molecular weight
markers (Sigma). 1 ml of a 0.5 mg/ml protein sample was applied to the
column, and each sample was chromatographed at 0.5 ml/min. An
additional study was conducted in which Inh was chromatographed at low
salt concentrations in the absence of glycerol. For this experiment, a
Bio-Gel P-100 (Bio-Rad) 150-ml column was equilibrated with 0.1 M NaCl TE (20 mM Tris-HCl, pH 7.9, 1 mM EDTA) and was calibrated with molecular weight markers
(Sigma). A 2-ml sample of 0.5 mg/ml Inh was chromatographed at 0.4 ml/min. Fractions were analyzed by Bradford assay, and logarithmic
plots of elution volume/void volume versus peak fraction were used for molecular weight calculations.
Protein-Protein Cross-linking--
9 µl of
His6-protein samples of Inh, Tnp, InhEQ451, or InhAD466,
0.1 mg/ml in 0.4 M NaCl HEG each, were mixed with 1 µl of freshly diluted 0.1% or 1% glutaraldehyde (Sigma). The reactions were
incubated at 25 °C for various times. 1 µl of 1 M
Tris-HCl, pH 8.0, was added along with 3 µl of SDS-PAGE loading
buffer, and the samples were incubated for 5 min at 68 °C. The
samples were analyzed by SDS-PAGE. The gels were stained with SYPRO
Orange (Bio-Rad) (analyzed by fluoroimaging) or Coomassie Blue.
GST-Tnp Pull-down Assays--
A GST-Tnp fusion protein was
overexpressed in E. coli DH5 Gel Shift Assay--
1 µl of His6-Inh,
His6-InhAD466, or His6-InhEQ451, 0.1 mg/ml
each, or 1 µl of a partially purified His6-Inh In Vitro Inhibition--
An in vitro assay for
Tn5 transposition has been developed which utilizes two
point mutations in Tnp, E54K and L372P, to promote both general and
trans activities of the otherwise inactive transposase in vitro (2), and we tested Inh for inhibitory activity in this system. The results of an in vitro transposition assay
performed in the presence of various concentrations of Inh are shown in Fig. 1. We show that the addition of
increasing amounts of Inh produces increasing inhibition. As the molar
ratio of Inh:Tnp approaches 1, transposition, as measured by donor
backbone excision, is near zero. Moreover, the inhibitory effect is
strong; 50% inhibition is achieved with a molar ratio of approximately
1:3 to 1:4, Inh:Tnp. We know that the inhibition is not the result of
nonspecific effects because a mutant Inh protein (InhAD466), added in
equal concentrations relative to Inh, was found to have no inhibitory
effect (this result is discussed below). If we assume that functional
synapsis is required for cleavage and that synapsis is the result of
one monomer of Tnp binding to each OE followed by Tnp-Tnp dimerization, then the inhibition experiment suggests inhibition before synapsis because one molecule is capable of inhibiting multiple molecules of
Tnp. For example, one Inh monomer should be sufficient to prevent the
formation of one synapse if it can heterodimerize with one Tnp monomer
that is bound to one transposon end, even if the other end is bound by
another Tnp monomer. This hypothesis assumes that the nonproductive
complex is relatively strong, and it does not rule out Inh-Tnp
interactions prior to Tnp-OE binding which might contribute to
inhibition.
Tnp or Inh Gel Filtration and Chemical Cross-linking--
The
potential for homodimerization of Inh or Tnp was addressed by both gel
filtration and covalent protein-protein cross-linking to probe the role
of multimerization in transposition or inhibition. The results of gel
filtration experiments conducted under various solution conditions are
presented in Table I. We have found that in the presence of glycerol (with buffer containing 0.1 M
NaCl) Inh can homodimerize, as has been shown previously (5). However, in the absence of glycerol (with buffer containing 0.1 M
NaCl) Inh is predominantly monomeric. At higher salt concentrations (with buffer containing 0.4 M NaCl and glycerol) Inh
homodimerizes, whereas Tnp is predominantly monomeric. Glutaraldeyde
cross-linking was used to confirm the homodimerization of Inh. Fig.
2 demonstrates that in the presence of
0.01% glutaraldehyde (with buffer containing 0.4 M NaCl
and glycerol) His6-Inh cross-links into homodimers, whereas
in 0.1% glutaraldehyde, His6-Inh cross-links into a
mixture of homodimers and higher order multimers (Fig. 2A,
lanes 3 and 5). Next, we tested
His6-Tnp in a glutaraldehyde cross-linking experiment and
found that, in contrast to His6-Inh, His6-Tnp
did not cross-link into dimers and remained relatively monomeric while exhibiting some aggregation, as deduced by the appearance of
His6-Tnp in the wells of the polyacrylamide gel (Fig.
2B). The relative instability of Tnp and its propensity for
aggregation have been observed by us and by others and correlates with
a tendency toward precipitation at lower salt concentrations. In
summary, both the gel filtration and cross-linking experiments indicate
that Inh homodimerizes whereas Tnp is monomeric. Furthermore, the
dimerization of Inh is facilitated by glycerol and is relatively
insensitive to salt, suggesting that hydrophobic effects may be
important at the dimer interface. Finally, because Inh lacks a short
part of the Tnp NH2 terminus, we speculate that the
NH2-terminal region of Tnp interferes with Inh-like
homodimerization.
GST-Tnp Pull-down Assay with Inh--
Interactions between Inh and
Tnp have been reported using a gel shift assay in which Tnp is bound to
one molecule of OE DNA (5, 6); here, we have sought any heterodimer
interactions that might occur in the absence of DNA. We used a
pull-down assay in which GST-Tnp was immobilized onto glutathione beads
before incubation with Inh. Then, the beads were washed and analyzed for retained protein. The assay was performed under the same conditions in which Inh is known to dimerize, whereas Tnp is monomeric (0.4 M NaCl with glycerol). Fig.
3A shows an SDS-PAGE analysis
of proteins recovered from this assay. We find that Inh is immobilized
only in the presence of GST-Tnp, and we conclude that Inh
heterodimerizes with Tnp.
Because two modes of heterodimerization have now been observed (free
and DNA-bound heterodimers), we repeated the pull-down assays to reveal
any differences between dimerization in the presence of OE DNA and
dimerization in the absence of DNA. To accomplish this, immobilized
GST-Tnp was incubated with or without a 3-fold molar excess of OE DNA
before the addition of 32P-labeled His6-Inh.
The amount of resulting immobilized 32P-labeled
His6-Inh was then measured and was found to be consistently higher in the presence of OE DNA (Fig. 3B) (variability was
observed between experiments probably because of differential washing
times; the error in independent experiments conducted in duplicate was 3%). Nonspecific DNA also increased the immobilization of
32P-labeled His6-Inh (data not shown) although
to a lesser extent than OE DNA. In summary, because more Inh was found
to bind to GST-Tnp in the presence of DNA, we hypothesize that the
DNA-bound heterodimer is relatively more stable than the free heterodimer.
OE DNA Gel Shift Assay--
To address whether Inh could
heterodimerize with TnpEK54/LP372 (the mutant protein used in the
in vitro transposition/inhibition assay), a gel mobility
shift experiment was performed in which Inh was added to Tnp-DNA
binding reactions (the Inh was added last, but the order of addition
has no effect on complex formation5
In such assays, Tnp exhibits little or no
monomeric binding, except in cases in which a COOH-terminal truncation
of Tnp is present (6, 8, 10). However, the addition of Inh is known to
promote strongly the binding of Tnp into a heterodimeric complex that
contains one DNA molecule (5, 6). In Fig.
4, the results of a gel shift assay with
radiolabeled OE DNA are presented. The heterodimeric complex bound to
one molecule of DNA is stimulated in the presence of the Inh fusion
protein (compare lanes 2 and 3, Fig.
4). This experiment confirms that Inh heterodimerizes with
TnpEK54/LP372 in a DNA-bound complex, and we suspect that this complex
may be important for inhibition. The addition of Inh also appears to
increase the complexes remaining in the well. This is a variable result
that is thought to be an artifact of Tnp aggregation.
InhEQ451--
A hypertransposing mutation in Tnp and Inh, E451Q,
has been identified previously in vivo (13) which, because
of its location near one of the known dimerization domains, was
predicted to function through a reduction in inhibitory regulation of
Tn5 transposition (9). We studied this mutation in the
context of His6-Inh for dimerization and for inhibitory
activity. Gel shift analysis with TnpEK54/LP372 (data not shown)
revealed that His6-InhEQ451 could heterodimerize with
Tnp-DNA as efficiently as His6-Inh. In addition, cross-linking experiments indicated that His6-InhEQ451
could homodimerize (Fig. 2A, lanes 4 and 6). Furthermore, His6-InhEQ451 was found to
inhibit TnpEK/LP in vitro to the same extent as
His6-Inh (data not shown). Thus, residue 451 is apparently
not involved in either Inh-like homodimerization, Inh-Tnp-DNA
heterodimerization or inhibition, as thought previously. However,
recent structural studies of Inh may explain why E451Q does not disrupt
dimerization. These studies reveal a COOH-terminal dimerization domain
in which Glu-451 is not part of the dimerization interface itself but
resides near the beginning of a long helix whose end forms the basis of
the interface (discussed further below).2 We hypothesize
that the E451Q mutation in Tnp influences transposition during some
step other than regulatory inhibition, perhaps during transposon DNA
end binding or during the capture of target DNA.
InhAD466--
We wished to mutagenize the COOH-terminal
dimerization domain of Inh at the recently discovered dimer
interface2 to test for functional effects. The dimer
interface is formed predominantly by the close interaction of two
helices in which the Inh Inh Inhibits Synapsis--
Here we find that purified Inh strongly
inhibits Tn5 transposition in vitro such that one
molecule of Inh apparently inhibits multiple molecules of
TnpEK54/LP372. This effect suggests that inhibition occurs prior to
synapsis because the present model of transposition requires two or
more functional Tnp molecules for every synapse, and therefore the
nonproductive complexation of one Tnp molecule at one transposon end
would block synapsis and effectively inactivate a second Tnp molecule
at the other end. Corresponding to our in vitro data, Inh
has been demonstrated to inhibit TnpEK/LP in vivo whereas
InhAD466 (see below) does not.6
DNA-bound Heterodimerization Is Likely the Predominant Inhibitory
Mechanism--
Inh and Tnp heterodimers were detected both free in
solution and within a DNA-bound complex, raising the possibility that either of these heterodimeric complexes may be important for
inhibition. Because the DNA-bound heterodimer is more abundant than the
free heterodimer in immobilization assays we suspect that the DNA-bound complex is highly stable. Correspondingly, we confirmed that Inh heterodimerizes with TnpEK54/LP372 in a gel shift experiment. Although
the shift experiment and our in vitro inhibition experiments were performed under different conditions using different OE DNA substrates (short OE-containing fragments and mutated OE-containing DNA
supercoiled circles, respectively), we are able to correlate DNA-bound
heterodimerization with inhibition. Further evidence that the DNA-bound
heterodimer is more stable than the free heterodimer has been found in
gel shift experiments in which the DNA-bound heterodimer product was
neither reduced nor enhanced by premixing Tnp and Inh.5
Therefore, it appears that monomers of Tnp bind to DNA, that heterodimerization occurs after DNA binding, and that free
heterodimerization has only minimal effects on DNA-bound heterodimer
complex formation. In summary, we suspect that DNA-bound heterodimers
may be the major mediators of inhibition.
In support of the hypothesis described above, we find that monomers of
Tnp are abundant. In gel filtration studies, Tnp was found to be
predominantly monomeric, whereas Inh could homodimerize. In addition,
no Tnp-Tnp dimers were detected in cross-linking studies although
Inh-Inh dimers were found. As discussed above, monomer-DNA binding is
probably prerequisite to dimer complex formation with Inh and could
also be important for synapsis, even though monomeric, untruncated Tnp
apparently binds slowly or with low affinity to OE DNA.
The COOH-terminal Domain of Inh/Tnp Is Critical for
Inhibition--
Mutation of the recently discovered dimerization
interface in Inh at the COOH terminus by site-directed mutagenesis
abolishes not only in vitro inhibition but also
homodimerization and OE-bound Tnp-Inh heterodimerization. We conclude
that this domain represents the critical dimerization main for
inhibition. The point mutation, A4660D, is not predicted to expose
unfavorable side chains to solvent upon disruption of the dimer
interface,2 and we correspondingly found that the mutant
protein was stable during overexpression and purification. Evidence
that the COOH-terminal region can be disrupted without disturbing other
functional regions in Inh is also found in our studies of Inh The NH2 and COOH Termini of Tnp and of Inh Are in Close
Proximity--
The COOH-terminal regulatory dimerization exerted by
Inh is a functional consequence of the deficiency of the first 55 amino acids of Tnp, perhaps uncovering the COOH-terminal domain and making it
available for dimerization. This hypothesis explains the monomeric
nature of Tnp in contrast to Inh and suggests that the NH2-
and COOH-terminal regions of Tnp are in close proximity or close
contact. In much the same manner, dominant-negative mutants of the
maize Activator transposase have been found by mutating the
NH2-terminal DNA binding region of the protein; these
mutants act by nonproductive oligomerization with functional
transposase (16). There could be a purpose for the interaction of
NH2 and COOH termini in Tn5 Tnp and, perhaps,
other transposases, at least in bacterial cells; usually the
NH2 terminus is associated with DNA binding, and because it
is produced first during translation it might bind to DNA before the
inhibitory COOH terminus can be produced. Perhaps such self-regulation
contributes to the cis phenotype of transposases in
prokaryotic cells, including that of Tn5, where
transcription and translation are coupled, and thus the nascent protein
is physically close to transposon end sequences. In fact, the COOH
terminus of Tn5 Tnp does appear to inhibit DNA binding
activity, as discussed below.
Interactions between NH2- and COOH-terminal regions
probably also occur in Inh, even though Inh is missing a short part of the corresponding Tnp NH2 terminus. When we removed 46 amino acids from the Inh COOH terminus we uncovered a secondary DNA
binding region that we suspect may be located near the NH2
terminus of the protein. In fact, structural studies show that the
NH2- and COOH-terminal regions are spatially close in a
three-dimensional structure of Inh,2 and nearby is a
disordered region containing a predicted helix-turn-helix, 107-124
amino acids.3 We can also expect the NH2
terminus of Tnp to be situated near this region, and it contains a
second predicted helix-turn-helix DNA binding region, amino acids
35-54.3 If this arrangement exists, it may resemble the
dual helix-turn-helix DNA binding motifs that have been identified in
protein-DNA structural studies of the Tc3 transposase (17) and the MuA
transposase (18).
Just as a COOH-terminal deletion uncovered a DNA binding function in
Inh, such deletions have also been found to stablize monomeric Tnp
binding to OE DNA (5, 7, 9), presumably via the known
NH2-terminal DNA binding domain. Thus, the COOH terminus of
Tnp partially interferes with DNA binding. Tnp-DNA binding is also
stabilized by heterodimerization with Inh (5), which could be caused by
removal of a hinderance of the COOH terminus via COOH-terminal
heterodimerization. A somewhat similar situation is found in the
Drosophila transposable P element system in which a KP
repressor, an internal deletion of transposase, binds DNA more readily
than the transposase and utilizes dimerization for high affinity
binding (19).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
EXPERIMENTAL PROCEDURES
70 °C.
430 (a COOH-terminal deletion), and Tnp using tailed EcoRI-containing primers
with Pfu polymerase (Stratagene) polymerase chain reactions
(9). The template for the InhEQ451 construct was pRZ622 (13). For InhAD466, the A466D mutation was introduced as a mismatch in one of the
primers. All oligonucleotides were synthesized by Research Genetics.
The polymerase chain reaction products were digested with
EcoRI and were ligated into the EcoRI site in
pET33b(+) (Novagen). The fusion proteins were overexpressed in E. coli and were purified as described previously (9).
His6-Inh, InhEQ451, and InhAD466 protein samples were
>95% pure based on Coomassie-stained SDS-polyacrylamide gels.
His6-Inh
430 was partially purified. The proteins were
stored in 0.4 M NaCl HEG (20 mM HEPES, pH 7.2, 1 mM EDTA, 10% glycerol) at
70 °C.
-mercaptoethanol, 2 mM spermidine, 100 µg/ml tRNA, 50 mM NaCl, 34 nM pGRST2 supercoiled plasmid DNA,
0.21 µM TnpEK54/LP372, and various concentrations of Inh,
His6-Inh, or His6-InhAD466. The reactions were
incubated at 37 °C for 1 h. XhoI (Promega) was
added, and the reactions were incubated further at 37 °C for 1 h. SDS was added to 0.5%, and the reactions were heated to 68 °C
for 15 min. Loading dyes were added, and the reactions were analyzed by
1% agarose gel electrophoresis in 1 × TAE electrophoresis
buffer. The gels were stained with SYBR Green (Molecular Probes) and
were analyzed by fluoroimaging.
using expression vector
pRZ47794 based on pGEX-2T
(Amersham Pharmacia Biotech). As a negative control, GST was expressed
from pGEX-2T. 1-liter cultures containing 100 mg/ml ampicillin were
grown at 37 °C to mid-log phase, and protein synthesis was induced
with 300 µM isopropyl
1-thio-
-D-galactopyranoside for 1.5 h. Cells were
harvested and resuspended in 10 ml of 0.3 M NaCl TEG and
were lysed using a French Press at 16,000 p.s.i. Lysates were cleared
by centrifugation. 0.5-ml samples of 50% glutathione-Sepharose 4B
beads (Amersham Pharmacia Biotech), equilibrated in 0.4 M
NaCl TEG, were added to 2.5 ml of the lysates, and the reactions were
incubated at 25 °C for 30 min. The beads were washed five times with
0.1% milk powder, 0.4 M NaCl TEG. 100 µl of 1.0 mg/ml
Inh was added to 50% bound-bead suspensions, and the reactions were
incubated at 30 °C for 30 min. The beads were washed with 0.4 M NaCl TEG, resuspended in SDS-PAGE loading buffer, and
boiled for 1 min. Supernatants were analyzed by SDS-PAGE, and the gels were stained with Coomassie Blue. For qualitative comparisons with and
without DNA, His6-Inh was labeled at the fusion kinase site
with [
-32P]ATP as described previously (9). 200 µl
of 50% suspensions of GST-Tnp-bound beads (approximately 5 µg/ml in
GST-Tnp) equilibrated in 0.2 M NaCl TEG was mixed with 90 µl of 200 mM potassium glutamate. 10 µl of a 29-bp
OE-containing DNA fragment (9) (0.2 mg/ml), calf thymus DNA (0.2 mg/ml), or H2O was added. 3 µl of 0.1 mg/ml 32P-labeled His6-Inh was added, and the
reactions were incubated at 25 °C for 1 h. 300 µl of 1% milk
powder, 0.2 M NaCl TEG was added. The beads were washed
twice with 0.2 M NaCl TEG, then they were resuspended with
100 µl of SDS-PAGE loading dyes and boiled for 1 min. Supernatants
were analyzed with 12% SDS-PAGE, and the gels were dried, imaged, and
quantitated by PhosphorImaging.
430
preparation (0.1 mg/ml in fusion protein) or mock preparation was
incubated in 20-µl reactions containing 0.7 µg/ml of a
32P-labeled, 60-bp, OE-containing DNA fragment (10) and 5 µg/ml Tnp or TnpEK54/LP372 in 10 mM HEPES, pH 7.2, 200 mM NaCl, 100 mM potassium glutamate, 0.5 mM EDTA, 5% glycerol at 30 °C for 30 min (final ratio
is 1:1 Inh:Tnp). 4 µl of loading dye was added, and the samples were
electrophoresed at 4 °C with 8% native PAGE in 1 × TBE
electrophoresis buffer. The gels were analyzed by autoradiography.
RESULTS
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Fig. 1.
In vitro inhibition of
transposition. Panel A, transposition reactions were
performed using a supercoiled plasmid DNA substrate and TnpEK54/LP372
with added Inh at various molar ratios, Inh:Tnp. After 1 h, the
reactions were digested with XhoI to linearize all DNA
molecules, and the reactions were analyzed using 1% agarose gel
electrophoresis and fluoroimaging of SYBR Green-stained gels, which
were quanititated. The full-length linear substrate and excised donor
backbone fragment are indicated, although other transposition products
are apparent at low molar ratios. Panel B, percent donor
backbone DNA, a measure of transposon double-ended cleavage, was
calculated, and the results were plotted against the molar ratio,
Inh:Tnp, for each lane in panel A. 50%
inhibition is taken as 18.5% donor backbone release because the
uninhibited reaction reached 37% donor backbone release.
Gel filtration results of Inh or Tnp analyzed under various conditions
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Fig. 2.
Glutaraldehyde protein-protein
cross-linking. Panel A, His6-Inh or
His6-InhEQ451 in 0.4 M NaCl HEPES buffer with
10% glycerol was reacted with 0.01 or 0.1% final concentrations of
glutaraldehyde for 5 h at 25 °C. The reactions were analyzed by
12% SDS-PAGE, and the gel was stained with Coomassie Blue. The
expected molecular masses of His6-Inh or
His6-InhEQ451 monomers and dimers are 52.0 and 104 kDa,
respectively. Panel B, His6-Tnp in
0.4 M NaCl HEPES buffer with 10% glycerol was reacted with
0.01% gluaraldehyde for various times, and the reactions were analyzed
by 9% SDS-PAGE. The gel was stained with Coomassie Blue. Expected
molecular masses are as follows: His6-Tnp, 57.8 kDa,
monomer, and 115.6 kDa, dimer.
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Fig. 3.
GST-Tnp pull-down assay for
heterodimerization. Panel A, GST-Tnp or GST,
immobilized on glutathione beads, was incubated with Inh, and the beads
were washed. Retained proteins were analyzed by 12% SDS-PAGE, and the
gel was stained with Coomassie Blue. Panel B,
GST-Tnp bound to glutathione beads was incubated with
32P-labeled His6-Inh with or without OE DNA,
and the beads were washed. Retained 32P-labeled
His6-Inh was analyzed by 12% SDS-PAGE; the gel was imaged
by PhosphorImaging and was quantitated.
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Fig. 4.
Gel shift assay; DNA-bound heterodimers.
His6-Inh or His6-InhAD466 was added to OE DNA
binding reactions in the presence or absence of TnpEK54/LP372; the
reactions were analyzed by 8% native PAGE and autoradiography.
-carbon of alanine 466 faces the interface,
adjacent to the corresponding
-carbon of residue 466 in the dimer
partner.2 Thus, the substitution of aspartic acid for
alanine at this position should disrupt the close interaction of the
dimer helices by steric occlusion and electrostatic repulsion. We
introduced the Ala
Asp mutation at position 466 by site-directed
mutagenesis and found the overexpressed His6-InhAD466
protein to be stable, as judged by relative expression levels and the
absence of detectable degradation fragments during purification (data
not shown). His6-InhAD466 was compared with
His6-Inh for inhibitory activity and, as shown in an
in vitro transposition assay in Fig.
5, was found to be inactive for
inhibition at all protein concentrations tested. We note that the
His6-Inh protein was slightly less active for inhibition
than Inh itself (this may reflect NH2-terminal fusion and
COOH-terminal domain interactions; the importance of the COOH terminus
for inhibition and evidence for the close proximity of NH2
and COOH termini is discussed below). Furthermore,
His6-InhAD466 did not homodimerize when tested in a
cross-linking experiment (Fig. 6), nor
did it heterodimerize in gel shift assays with DNA-bound Tnp (data not
shown) or TnpEK54/LP372 (Fig. 4, lane 4), in
contrast to His6-Inh. We conclude that the A466D mutation
disrupts the dimer interface at the COOH terminus of the protein and
that this interface is critical for inhibition.
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Fig. 5.
In vitro inhibition of
transposition. His6-Inh or His6-InhAD466
was added to transposition reactions at molar ratios of 0, 0.5, or 1.0 His6-protein:TnpEK54/LP372. The reactions were digested
with XhoI and were analyzed using 1% agarose gel
electrophoresis with ethidium bromide staining. The inhibitory effect
of His6-Inh is slightly reduced compared with Inh as in
Fig. 1.
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Fig. 6.
Glutaraldehyde protein-protein
cross-linking. His6-Inh or His6-InhAD466
was cross-linked for various times in 0.01% glutaraldehyde. The
reactions were analyzed by 9% SDS-PAGE; the gels were stained with
SYPRO Orange (Bio-Rad) and were analyzed by fluoroimaging.
430--
To study further the effects of mutating the COOH
terminus of Inh, a truncation was introduced which deletes all residues COOH-terminal to residue 430. His6-Inh
430 was found to
have relatively low expression levels in E. coli compared
with His6-Inh. We tested partially purified preparations in
DNA binding assays (in the absence of Tnp). Because Inh has not been
previously reported to bind to DNA, we were surprised to find that
His6-Inh
430 could bind to OE DNA (Fig.
7). Mock preparations did not show OE DNA binding activity (data not shown), nor did His6-Inh (Fig.
7). The shifted complex consists of a bound His6-Inh
430
monomer, as judged by comparison with the monomeric binding of a
previously characterized protein, Tnp
387 (6, 10) (data not shown). Thus, the COOH-terminal deletion in Inh has apparently exposed a
previously unknown DNA binding region.
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Fig. 7.
Gel shift assay; Inh truncation.
His6-Inh or His6-Inh 430 was incubated with
OE DNA, and the reactions were analyzed by 8% native PAGE. The gel was
imaged by autoradiography.
DISCUSSION
430
because this protein can bind to DNA. A second COOH-terminal mutated
protein, InhEQ451, was found to be active for inhibition and could
homodimerize and also heterodimerize with OE-bound Tnp. Although
residue 451 is located near the COOH-terminal dimerization domain, this
result confirms that the critical dimer interface is located at the end of the long COOH-terminal helices of the dimer.2 In
summary, Inh COOH-terminal homodimerization reflects the mechanism of
Tn5 inhibition, and this mechanism likely occurs through Inh heterodimerization with Tnp, probably via DNA-bound complexes.
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ACKNOWLEDGEMENTS |
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We are indebted to Dona York and to Mindy Steiniger-White for sharing unpublished data and for many helpful discussions. We thank Michael Weinreich for the GST fusion construct. We gratefully acknowledge collaborators Ivan Rayment, Hazel Holden, and Doug Davies for the structural work on the Inh protein that we have discussed in this paper. Finally, we thank A. S. Silbergleit, V. A. Lanzov, and N. Benuch for communicating secondary structure predictions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM50692.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.
§ Predoctoral trainee on National Institutes of Health Training Grant GM07215.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, 433 Babcock Dr., University of Wisconsin-Madison, Madison, WI 53706. Tel.: 608-262-3608; Fax: 608-262-3453; E-mail: reznikoff{at}biochem.wisc.edu.
2 D. R. Davies, L. A. M. Braam, H. Holden, W. S. Reznikoff, and I. Rayment, in preparation.
3 A. S. Silbergleit, V. A. Lanzov, and N. Benuch, personal communication.
4 Constructed by M. D. Weinreich.
5 D. York and W. S. Reznikoff, unpublished data.
6 M. Steiniger-White, L. A. M. Braam, and W. S. Reznikoff, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: Tnp, transposase; bp, base pair(s); OE, transposon outside end(s); Inh, transposase inhibitor; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase..
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REFERENCES |
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