Analysis of tet operator-TET repressor complexes by thermal ...
Transcript of Analysis of tet operator-TET repressor complexes by thermal ...
volume 10 Number 19 1982 Nucleic Acids Research
Analysis of tet operator-TET repressor complexes by thermal denaturation studies
Wolfgang Hillen, Bemhard Unger and Gerd Klock
Institut fur Organische Chemie und Biochemie, Technische Hochschule Darmstadt, Petersenstr. 22,D-6100 Darmstadt, FRG
Received 6 August 1982; Accepted 13 September 1982
ABSTRACT
Interaction of the Tn10 encoded TET repressor with the tet op-erator is studied by thermal denaturation of the specific com-plexes employing operator containing purified DNA restrictionfragments varying in length from 187 bp to 501 bp. Comparison ofthe melting curves obtained with the free DNA and DNA-repressorcomplexes revealed a specific stabilisation of the operator con-taining cooperatively melting segment in multiphasic denaturationcurves. Under limiting concentrations of TET repressor the dena-turation of the free DNA is observed next to the denaturation ofthe repressor•DNA complex. Quantitative analysis yields a bindingcurve with a stoichiometry of four TET repressors per tet oper-ator containing fragment. The denaturation temperature of thecomplex is almost independent of the ionic strength indicatingthat the protein component denatures at this temperature. Thehalf life time of the TET repressor•tet operator complex isgreater than 100 min under these conditions. The tet operator onthe 187 bp fragment is determined to be located between a Xba Iand a Sau 3a site by removing base pairs from either end of thefragment and subsequent comparison of the melting curves. It isconcluded that the TET repressor recognizes the double strandedrather than a possible cruciform structure of the tet operator.The influence of a regulatory protein on the thermal stabilityof a genetic control region is discussed with respect to itspossible influence on the initiation of transcription.
INTRODUCTION
DNA binding proteins may preferentially form complexes with
either the double stranded or the single stranded structure of
DNA [1,2]. Helix destabilising proteins e.g. lower the T of
DNA due to their preferential binding to single strands which is
thought to aid replication [3-6]. On the other hand, histones
recognize the double stranded structure of DNA which results in
an increased T m of the core particle as compared to the free
DNA [7]. These interactions are non-specific with regard to the
DNA sequence.
© IR L Prats Limited, Oxford, England. 60850305-1048/82/1019-6085S 2.00/D
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Proteins active in regulation of gene expression recognize
specific binding sequences [1] with an increased association
constant over non-specific interactions with DNA [8]. Their ef-
fect on the thermal stability of the DNA has been described so
far only for the non-specific interaction t£rl£4. The study of
specific effects requires the availability of the target DNA
and the respective protein in large amounts. Furthermore, the
thermal stability of the protein must extent to higher temper-
atures than the T of the DNA binding sequence. This can be
achieved by low salt concentrations because the stability of the
protein is less sensitive to the ionic strength than the Tffl of
the DNA [9-11].
Recently we compared the thermodynamic and genetic proper-
ties of the tet gene control region located on the transposon
Tn10 and demonstrated a specific stabilisation of the tet oper-
ator upon complex formation with the TET repressor [12]. In this
article we describe a detailed experimental analysis of the
thermal stability of TET repressor•tet operator complexes using
small, purified DNA restriction fragments containing the tet
operator.
MATERIALS AND METHODS
General Methods
Restriction endonucleases were purchased from BRL, Bethesda,
Md or Boehringer, Mannheim. Eco RI was prepared as described
[13]. TET repressor protein was isolated from E. coli MO trans-
formed with pRT211 [14] as described [15]. The DNA fragments
were prepared from pWH106, pWH122, and pWH141 [15] and purified
by RPC-5 chromatography [16,17].
Optical Measurements
The samples were prepared for the melting experiments essen-
tially as published previously [12] except that the TET repressor
was added after degassing with helium. The amount of TET repres-
sor stock solution added did not exceed 10% of the total volume
and was usually around 3%. No change in the optical density was
observed in control experiments with the protein alone. Collec-
tion and handling of the data was as published [12]. After the
experiments the nucleic acids were routinely analyzed by poly-
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aorylamide gel electrophoresis to confirm the absence of any de-
gradative activity. The ionic strength of the samples was in all
cases 5 mM sodium cacodylate, pH 7.0, 0.1 mM EDTA, and 4 mM so-
dium chloride unless stated otherwise in the legend to the respec-
tive figure.
RESULTS
Description of the genetic system
The DNA fragments used in this study are outlined in figure 1
with respect to the location of their genetic functions [15].
The tetracycline resistance mediating tet gene from the trans-
poson Tn10 consists of an overlapping bidirectional promotor•op-
erator system [14,15,181. The TET repressor regulates trans-
cription of the TET protein as well as it's own synthesis. Se-
quence analysis of the regulatory region suggests the existance
of two potential operators [19]. The expression of the TET - and
TET repressor genes is inducible by tetracycline as indicated in
figure 1.
We confirmed previously that the TET repressor specifically
Figure 1 Genetic and physical description of the DNA fragmentsused in this study. The top panel describes the regulatory func-tions involved with expression of the Tn10 encoded tetracyclineresistance. The synthesis of two mRNAs starts from an overlappingPribnow box in both directions. The TET repressor is drawn as abox in the tet operator binding structure and as a circle afterthis function has been inactivated by tetracycline. H denotes aHinc II -, X a Xba I -, and S a Sau 3a site. The latter two areused to remove base pairs from the end of the 187 bp DNA in orderto locate the tet operator region (see [15] and references citedtherein for a detailed description).
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Figure 2 Thermal denatura-tion curves of the 501 bpfragment. B: Thermal dena-turation curve of the freeDNA. A: Melting curve of the501 bp DNA in the presenceof a fourfold molar excessof TET repressor offset by0.6 on the vertical scale.The salt conditions are:4 mM sodium chloride, 5 mMsodium cacodylate, pH 7.0,and 0.1 mM EDTA.
60 62 6t 66
stabilizes a cooperative segment of a 1450 bp long DNA fragment
containing the tet operator. In order to study this stabilisation
we used smaller DNA fragments containing the tet operator to in-
crease the relative amount of target sequence over flanking DNA.
The smallest of the DNA fragments outlined in figure 1, which is
187 bp long, is then used to further characterize the TET repres-
sor- tet operator complex. The location of the tet operator on the
187 bp fragment is determined to be on roughly 100 bp between the
Xba I - and Sau 3a sites indicated in figure 1.
Thermal denaturation of TET repressor-tet operator complexes
Figure 2 displays the melting curves of the 501 bp fragment
alone and with a fourfold molar excess of TET repressor. Figure 3
shows the thermal denaturation of the 461 bp DNA with and without
Figure 3 Thermal denaturationcurves of the 461 bp fragment. Themelting curves are shown for theDNA alone (solid line) and in thepresence of a fourfold molar excessof TET repressor (dashed line). Thelatter curve is offset by 0.4 onthe vertical scale. The saltconditions are the same thanin figure 2.
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61 65
TBHPERATURECC)
Figure 4 Melting curves of the187 bp DNA. The thermal denatu-ration of the free DNA (solid line)and in the presence of a fourfoldmolar excess of TET repressor(dashed line) is shown. The saltconditions are as given infigure 2.
a fourfold molar excess of TET repressor and figure 4 gives the
results of the same experiments with the 187 bp DNA. All three
results demonstrate a specific stabilizing effect of the TET re-
pressor on the cooperatively melting sequence containing the
tet operator. The T - and area analyses of these data is summa-
rized in table I. The 501 bp fragment in figure 2 denatures in
three clearly separated transitions [12]. Addition of the TET
Table
501
Tma [°
60.7
63.6
64.6
187
Tm f ° C
56.8
I Tffl and
bp
C] b Pb
175
110
216
bp
] bp
187
area analysis of
501 bp
Tm t"C
64.6
66.0
66.8
187 bp
Tm f ° C ]
62.0
67.5
TETR c
bp
138
286
74
TETR
bp
73
114
the melting
461 bp
Tm ro
59.3
66.0
-
curves
bp
150
311
-
148d-TETR
Tm ro
62.5
67.3
bp
30
118
461
Tm [
61.1
67.2
68.6
139e
Tm [
60.8
65.8
bp•TETR
°C] bp
68
319
74
bp-TETR
°C] bp
79
60
a Accuracy of T determination is ±0.5°Cb Accuracy of tne area analysis is ±10%c TET is the abbreviation for a fourfold molar excess of TET
repressord from the Xba I digeste from the Sau 3a digest
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repressor stabilizes the one with the lowest T by about 5°C. It
is also apparently split into at least two cooperatively melting
sequences. Due to the limited resolution of the experiment it is
not possible to clearly describe the changes in the melting pro-
file ±n -this case. - - -
The 461 bp fragment denatures also in two subtransitions
(Figure 3) with the least stable one containing the tet operator
[12]. In the melting curve of the 461 bp-TET repressor complex
this cooperative unit is split into two new transitions. One is
broadened and shifted only slightly to higher temperatures and
the second is stabilized by about 9°C and appears to be more
stable than the sequence of the main transition. Figures 2 and 3
reveal that also the melting transitions of sequences lacking
the tet operator are stabilized. The increase in T is, however,
only 1°C which is probably the result of small changes in the
ionic strength brought about by the addition of TET repressor
stock solution as discussed below.
The observations from the 461 bp DNA are confirmed by the
melting of the 187 bp fragment. The single transition in fig-
ure 4 results from the same sequence than the early transition
in figure 3 (compare figure 1) plus additional 35 bp. Upon sat-
uration with TET repressor the single cooperative unit of the
187 bp DNA is split into two cooperative units which are stabi-
lized to a different extend. The T m of the first is shifted by
about 5°C and the one of the second by about 11°C. This result
is very similar to the one observed for the early transition of
the 461 bp DNA in figure 3.
Effect of TET repressor concentration on the thermal denatura-
tion of the 187 bp DNA
Figure 5 demonstrates the effect of increasing TET repressor
concentration on the melting of the 187 bp DNA. As the molar
ratio of TET repressor over tet operator increases the area of
the single transition decreases whereas the areas of the newly
formed transitions increase. Also, the Tffl of the single transi-
tion is shifted towards higher temperatures. The interpretation
of this result iB: First, as the TET repressor concentration is
increased more of the 187 bp fragment is converted to the TET
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Figure 5 Thermal denaturationof the 187 bp fragment in thepresence of various amounts ofTET repressor. The molar ratioof TET repressor to DNA andthe offset on the verticalscale are: A fourfold, 1.7;B threefold, 1.4; C twofold,1.0; D equimolar.
61 65IBHRAIUKIT]
repressor-tet operator complex. As a result two different mole-
cular species are melting in those experiments. The sharp tran-
sition reflects the thermal denaturation of the remaining free
187 bp DNA, and the two other transitions result from the mel-
ting of the 187 bp*TET repressor complex. The latter two tran-
sitions are, thus, the only remaining transitions when the 187 bp
DNA is saturated with TET repressor (Figure 4). Secondly, the
T m of the 187 bp DNA is increased because salt is added with the
TET repressor stock solution. Due to the low ionic strength of
4 mH in these experiments the effect on the T is rather large.
An alternative explanation could be the non-specific binding of
TET repressor to the 187 bp fragment. The latter is ruled out by
an analysis of the areas under the melting transitions. The plot
of bound DNA as determined from the area of the complex denatura-
tion versus the total TET repressor concentration yields a titra-
tion curve which agrees quantitatively with the one obtained from
nitrocellulose filter binding [12,15]. This analysis confirms
that the specificity of binding is the same in these experiments
than in the filter binding studies, where specificity could be
demonstrated by the lack of competition by other DNA fragments
[15].
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Effect of ionic strength on the melting of a TET repressor•tet
operator mixture
Figure 6 shows the melting of the 187 bp DNA in the presence
of two moles TET repressor at different ionic strengths. Whereas
the Tm of ~llie TET repressorriret operator complex ±s almost inde-
pendent of the ionic strength the T of the free DNA depends
strongly on the salt concentration as expected [21]. At 40 mM
NaCl the Tms overlap and at higher ionic strengths the TET re-
pressor • tet operator complex denatures to yield the double
stranded free DNA. Therefore, this denaturation is not observed
in a melting experiment. It may be concluded that the TET re-
pressor • tet operator complex is stable up to 69°C which agrees
satisfactorily with the value of 65°C previously reported for
higher ionic strength [15].
Location of the TET repressor binding site on the 187 bp DNA
According to previous results the TET repressor binds to se-
quences located to the right of the Xba I site as drawn in fig-
ure 1 [15]. In order to determine the location of the DNA melting
under the two transitions in the 187 bp-TET repressor complex
the 187 bp DNA was digested with Xba I to remove sequences from
the left end and with Sau 3a to remove sequences from the right
Figure 6 Melting curve of the187 bp DNA with a twofold molarexcess TET repressor at differentsalt concentration. All experi-ments are done with 5 mM sodiumcacodylate, pH 7.0, 0.1 mM EDTA.The sodium chlorideconcentrations and offsets onthe vertical scale arei A 36 mM,1.3; B 27 mM, 0.8; C 18 mM, 0.4;D 4 mM.
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end as drawn in figure 1. The melting curves of the resulting
purified fragments complexed with TET repressor are shown in
figure 7. This result demonstrates clearly that the first tran-
sition results from sequences located to the left of the Xba I
site, whereas the second transition is caused by sequences to
the right of approximately the Hinc II site complexed to the
TET repressor. The formation of the TET repressor-tet operator
complex is not affected by the lack of about 50 bp [14] from
the right end of the 187 bp DNA. Figure 7 reveals, however,
that the number of base pairs melting under transition two is
reduced in this case. The T - and area analyses from these re-
sults are summarized in table I.
The physical description of changes in the thermal denatura-
tion of the 187 bp fragment upon TET repressor binding is, thus,
that the cooperative unit is destroyed by the TET repressor
binding near the Hinc II site acting as a clamp to keep the
double strand together. Sequences to the left of the Xba I site
in figure 1 denature at elevated temperatures because they can-
not dissociate anymore. The tet operator sequence together with
base pairs to the right of it denature at the Tffl of the TET re-
pressor • tet operator complex. This analysis agrees well with
the notion that the sequences to the left of the Hinc II site
are more AT rich than the ones to the right of the Hinc II site
[19]. Also, the analysis of the 461 bp denaturation reveals that
the rightmost sequences of the 187 bp DNA embodied in this frag-
ment are part of a more stable cooperative unit [12].
Figure 7 Melting curves ofsubfragments of the 187 bp DNAwith a fourfold molar excess ofTET repressor. A: The leftmost38 bp were removed by Xba Idigestion and the curve isoffset by 0.8 on the verticalaxis. B: The rightmost 40 bpwere removed by Sau 3adigestion. The curve is offsetby 0.4 on the vertical scale.The salt conditions are asdescribed in figure 2.
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DISCUSSION
Figures 2-4 demonstrate clearly the effect of TET repressor•
tet operator complex formation on the thermal denaturation pro-
file of the respective DNA fragment. These results together with
the previously described effect on a 1450 bp DNA [12] indicate
specific binding of the TET repressor to the tet operator under
these conditions. Specificity of binding is concluded from the
particular stabilisation of the tet operator containing coopera-
tively melting segment of the fragments in comparison to a lesser
extend of stabilisation of the non-specific sequences. Additional
evidence for specificity comes from control experiments employing
DNA fragments lacking the tet operator. In these cases only the
slight increase in Tffl was observed very similar to the results
described in figures 2 and 3 for the non-specific DNA segments
[12]. Specificity of binding is most convincingly demonstrated
by the effect of the TET repressor on the 187 bp fragment. Be-
cause the free DNA denatures in one single cooperative transi-
tion non-specific binding of the protein could very well alter
the T of the DNA, but never disrupt the cooperativity of the
denaturation. This observation can only be explained when the
TET repressor binds to a specific part of the fragment only. If
the stabilisation of this segment is large enough the cooperati-
vity may be disrupted as shown in figure 4 for the 187 bp-TET
repressor complex. The not occupied segment of the fragment is
also stabilized because the single strands cannot dissociate
anymore. It has been shown that the data in figure 5 yield a
titration curve when analyzed for the amounts of free and com-
plexed DNA [20] . This binding curve agrees quantitatively with
the one obtained from nitrocellulose filter binding [15]. Taken
together, these results may be interpreted as i) specific
binding of the TET repressor to the tet operator under these
conditions and ii) a stabilisation of the double stranded opera-
tor structure in the TET repressor.tet operator complex. It seems,
therefore, rather unlikely that cruciform structures are involved
in operator recognition in this case.
Non-specific binding of lac repressor [9] and CRP [10] to DNA
stabilizes the double stranded structure as indicated by an in-
crease in Tm> Specific studies require that the thermal stability
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of the protein-DNA complex exceeds the one of the free DNA. This
can only be achieved if the target DNA sequence is AT rich [11]
and the ionic strength of the experiment is low [21]. In this
case the ionic strength must be below 40 mM (Figure 6) and is
about 4 mM NaCl in the other experiments. It has been shown that
the non-specific association constant of the lac repressor to
DNA is increased with decreasing ionic strength [22] . The results
of this study show that the specificity of binding of the TET
repressor extends down to 4 mM NaCl.
The half life time of the TET repressor-tet operator complex
must be greater than 100 min under the conditions of the melting
experiment in figure 4 since it takes about 100 min to raise the
temperature from the T of the free DNA to the T of the complex.
During that time any dissociation would result in the subsequent
denaturation of the free DNA and probably also of the TET re-
pressor [15].
The T m of the repressor-operator complex is nearly indepen-
dent of the ionic strength as shown in figure 6. It is, there-
fore, likely that the T reflects the denaturation temperature
of the protein component in the complex, which is irreversible
on the time scale of the melting experiment.
The location of the operator on the 187 bp fragment is ex-
perimentally determined by making use of the restriction sites
on this DNA. As shown clearly in figure 7 the upstream part
(as seen from the tet gene) is not involved with repressor
binding. The conclusion, therefore, is that the operator is be-
tween the Xba I and Sau 3a sites. This agrees well with previous
results from protection experiments [15] and with the location
of possible operators deduced from the DNA sequence [19].
The thermal stability of two DNA segments active in gene re-
gulation studied so far show a clear correlation of the genetic
and thermodynamic properties. In the E. coli lactose genetic
control region the CRP and RNA polymerase binding sites are lo-
cated within the same small cooperatively denaturing segment
[23,24]. The lac operator sequence forms the boundary of this
DNA segment. Therefore, all three proteins may alter the stabili-
ty of the double stranded structure of this region. Unfortunate-
ly the results published so far describe only non-specific bind-
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ing influences of the lac repressor [9] and CRP [10] on the
thermal stability of DNA.
A similar correlation of genetic and thermodynamic properties
has recently been described for the Tn10 encoded tetracycline
resistance genetic control region IT2T." The promotor arid'Opera-
tor sequences are within an unstable segment which is 140 bp
long. In this case the effect of specific binding of the TET re-
pressor to the tet operator is a stabilisation of the double
stranded structure of the operator. Thus, the repressor counter-
acts the RNA polymerase which is known to bind to thermally un-
stable regions [25,26] and unwind about ten bp upon complex for-
mation [27]. It appears to be possible that the thermal stability
of a regulatory region influences the initiation rate of tran-
scription off this promotor. In that case a repressor would be
expected to act as demonstrated for the TET repressor in this
study, whereas an activator protein should exhibit a contrary
influence. Non-specific binding of CRP to DNA increases the Tffl
[10]. However, the specific influence of CRP on the lac regulato-
ry region remains to be measured. A possible decrease of T m of
the lac control region upon binding of CRP could lend additional
support to this speculation.
ACKNOWLEDGEMENTS
We wish to thank Dr. H.G. Gassen for many fruitful discus-
sions and Mrs. E. ROnnfeldt for her help preparing the manu-
script. This work was supported by the Deutsche Forschungsge-
raeinschaft and by ROhm GmbH, Darmstadt.
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