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.

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Nucleic Acids Research

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-



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



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.

6092



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

6094



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-

6096



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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Analysis of tet operator-TET repressor complexes by thermal