1 Department of Biochemistry and 2 Department of Biophysics, Bose Institute, Acharya J. C. Bose Birth Centenary Building, P 1/12 C.I.T Scheme VII M, Calcutta 700 054, India
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Abstract |
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Keywords: cooperativity/free energy/loop/repressor/transcription
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Introduction |
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Owing to the torsional rigidity of DNA, not all DNA-bound proteins are able to interact with each other. This has been clearly demonstrated in many systems where two otherwise interacting proteins are placed on the opposite faces of the DNA (Hochschild and Ptashne, 1986; Bandyopadhyay et al., 1996
). Clearly, the orientation of the interacting patches of the two proteins and the energy required to bring them to juxtaposition are crucial to interaction between the two proteins and loop formation. This concept is the basis of the differential contact model of transcription regulation (Roy et al., 1998
). Hence a more quantitative understanding of the energetics involved in proteinprotein interaction while bound to DNA is essential.
Lyticlysogenic switch of bacteriophage has emerged as one of the best characterized systems for the study of proteinprotein interactions that regulate transcription (Ptashne, 1992
). In this regulatory system,
-repressor binds to several pairs of operator sites (e.g. OR1OR2, OL1OL2) with concomitant interaction between two adjacent site bound dimers. Primarily due to work of Ackers and co-workers (Senear et al., 1986
) we have obtained an estimate of the interaction energy, which is ~23 kcal/mol. This interaction energy is a net result of several distinct processes. In this work we have attempted to dissect the cooperative interaction into component processes which can then be related to structural changes. More importantly, we have attempted to estimate these various components of the interaction energy using a
-repressor mutant, S228N, that is defective in higher order aggregate formation (Burz and Ackers, 1994
; Burz et al., 1994
), but not in interaction between two dimers bound to adjacent operator sites.
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Materials and methods |
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c47 (cI) phage and pEA305 plasmid were obtained from Professors M.Lieb and M.Ptashne, respectively (Amman et al., 1983). DTNB, acrylamide, IPTG, PMSF, BSA, polyethylenimine, DTT, calf thymus DNA, DNase and RNase, ß-mercaptoethanol and glycerol were purchased from Sigma Chemical (St. Louis, MO). Restriction enzymes were obtained from GENIE (Bangalore India). Bacto-Agar, bacto-tryptone and yeast extract were supplied by Difco (Detroit, MI). All other reagents were of analytical grade and were procured from local suppliers.
Cloning of the cI gene of carrying c47 mutation
The cIc47 mutant gene (same as S228N mutant repressor) of was cloned by ligating the 4.679 kb EcoRI/BamHI fragment of
c47 DNA with the 3.987 kb EcoRI/BamHI fragment of pBR322 to obtain the plasmid pMJ47. The cI gene in this plasmid was sequenced fully using suitably designed primers. A cassette plasmid pMS1 containing the wild-type cI gene under the control of tac promoter and having a ClaI site between the C-terminus of cI gene and the downstream HindIII site was constructed earlier (Das and Mandal, 1986
). The S228N mutant cI gene was subcloned in pMS1 by exchanging its 803 bp DNA segment bounded by the C-terminal-upstream NsiI and downstream ClaI sites with the same NsiIClaI DNA fragment from the above pMJ47 to obtain pMSJ47 carrying the S228N mutant cI gene under the control of tac promoter.
Repressor purification
The S228N mutant repressor was purified from E.coli RR1 (15 lacZ) carrying the plasmid pMSJ47 by the procedure given by Saha et al. (1992). The concentration of native repressor was determined using the relation E1% = 11.3 and was calculated in terms of monomer unless stated otherwise. For all studies, the repressor was dialyzed against 0.1 M phosphate buffer, pH 8.0.
Chemical modification
The repressor was dansyl labeled by treatment with a 10-fold molar excess of dansyl chloride according to Banik et al. (1993). Sulfhydryl reactivities were measured using DTNB as described by Banik et al. (1992).
Fluorescence methods
All fluorescence spectra were measured with a Hitachi F 3000 spectrofluorimeter with a computer for spectra addition and subtraction facility. For tryptophan fluorescence, the excitation and emission wavelengths were kept at 295 and 340 nm, respectively, and the inner filter effect correction was made according to Bandyopadhyay et al. (1995). Anisotropy was measured using a Hitachi polarization accessory as described by Banik et al. (1993). Acrylamide quenching methods were also described by Bandyopadhyay et al. (1995).
CD spectra
Far-UV circular dichroism (CD) spectra were measured in a JASCO J600 spectropolarimeter. The CD spectra were measured in a 1 or 10 mm pathlength cuvette as required, at ambient temperature, controlled at 25°C. The scan speed was 50 nm/min and 10 scans were signal averaged to increase the signal-to-noise ratio. This study was made in 0.1 M phosphate buffer, pH 8.0.
Denaturation study
A series of freshly prepared solutions of ultrapure urea having 0.510 M concentrations in 0.1 M potassium phosphate buffer (pH 8) were prepared and S228N repressor was added to a final concentration of 1 µM. Equal volumes of buffer were added to the same volume of urea solution and these mixtures were used as a blank. Tryptophan fluorescence intensity was monitored at 340 and 350 nm and the same experiment was repeated with wild type -repressor and compared.
Tetramerdimer dissociation in the presence of OR1
Dansyl-labeled S228N repressor was mixed with unlabeled S228N repressor at a ratio of 1:9 to achieve a high concentration (~30 µM). It was then mixed with a stoichiometric amount of single operator OR1 so that final concentration of the operator was 15 µM, to form operator-bound S228N complex. The anisotropy value of this complex was measured. It was then progressively diluted with 0.1 M potassium phosphate buffer, pH 8.0, and anisotropy values at different protein concentrations were determined. In all cases the buffer blank value was subtracted from each experimental value. The excitation wavelength was 340 nm and the emission wavelength was 520 nm. A computer averaging transient scan was performed to increase the signal-to-noise ratio.
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Curve fitting |
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where Aobs is the observed anisotropy, Am is the anisotropy of the monomer, Ad is the anisotropy of the dimer, Kd is the dissociation constant and [P] is the total monomer concentration.
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Results |
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The -repressor dimer binds to pairs of operator sites with concomitant interaction between the dimers resulting in cooperative binding. This cooperatively bound complex is accompanied by DNA and protein distortion. Figure 1
shows the deconstruction of the cooperative complex formation in terms of steps that can be estimated and related to structural changes. Clearly, the net cooperative interaction energy (
Gcoop) is sum of the interaction energy between two repressor dimers bound to two isolated operators (
Gint), loss of rotational and translational entropy by being associated with the same DNA molecule (as opposed to two separate DNA molecules) (
Gprox) and the proteinDNA distortion energy needed to bring the two interacting protein surfaces in juxtaposition (
Gdis):
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From an experimental point of view, the exact equivalence of the two sides is not possible as Gint can only be measured for two repressors bound to identical operator sites and not to different operator sites, e.g. OR1 and OR3. As has been mentioned above,
Gcoop has been estimated for
-repressor cooperative interaction. Thus, measurement of any two quantities on the right-hand side of the equation should be sufficient to arrive at an estimate of all three quantities. Both
Gprox and
Gdis are difficult to measure experimentally. As will be described below, however, the magnitude of
Gprox can be estimated based on experiments done on other systems. Thus an estimate of
Gint should provide an estimate of energies of steps leading to cooperative complex formation.
We have already reported a method for measuring the interaction energy of two repressor dimers bound to an oligonucleotide containing an operator site (Banik et al., 1993). Owing to operator-induced conformational changes in the repressor and consequent modulation of the nature of the cooperative interaction energy (Bandyopadhyay et al., 1996
), it is necessary that the interaction energy be measured between repressors bound to isolated operator sites. However, such a measure of interaction energy cannot be taken as a measure of
Gint without additional evidence. It needs to be established that the interface between the two isolated operator-bound repressors is the same as the interface involved in the cooperative interaction.
Ackers and co-workers have demonstrated that S228N mutant of -repressor retains full cooperative interaction energy, but at the same time loses much of the ability to form free tetramers in solution (Burz and Ackers, 1994
; Burz et al., 1994
). In addition, S228N mutant is defective in monomerdimer association (see later) (Burz and Ackers, 1994
). Despite the weakened monomerdimer association, the binding of S228N to the OR1 operator site is complete below 1 µM protein concentration (Burz and Ackers, 1994
). The dimeroperator interaction energy is sufficient to overcome the monomerdimer association defect in this concentration range. Hence it is possible to measure the interaction between two S228N dimers complexed with operator site OR1 by measuring self-association of the complex at concentrations above 1 µM. Figure 2
shows the fluorescence anisotropy of dansyl-labeled S228N
-repressorOR1 complex as a function of complex concentration. The anisotropy increases as a function of complex concentration, suggesting association between two dimers. A fit to the appropriate equation (see Materials and methods) yields a dissociation constant of 6 µM. Thus, although this mutant has lost its ability to tetramerize in the free state, like the cooperative interaction, it has largely retained the interacting ability while operator bound. This strongly suggests that the interface involved in the cooperative interaction is similar to, if not identical with, that of the repressors bound to two isolated operator sites.
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The similar association of properties of S228N repressor while bound to a single operator site suggests that such an interaction energy may be interpreted as Gint. It would be wise, however, to make such an interpretation only if the structure of the mutant repressor remains similar to that of the wild-type. Mutations are known that cause phenotypic changes indirectly by causing structural perturbations (Deb et al., 1998
). Such structural perturbations may lead to new interaction surfaces unrelated to the native one. We therefore explored the structure of the S228N repressor with various spectroscopic and biochemical tools. Figure 4A
shows that the CD spectra of both wild-type and the S228N repressor at 0.5 µM concentration are virtually identical. Since, at this concentration, the wild-type protein is dimeric and the S228N mutant repressor is monomeric, the above result suggests that very little change occurs in the secondary structure following dimer formation. Figure 4
(B) shows the CD spectra of both the wild-type and S228N repressors at 15 µM concentration. At this higher concentration, the wild-type repressor is predominantly in the tetrameric state (Banik et al., 1993
), whereas the S228N mutant repressor is present in the dimeric state along with a significant fraction of the population in the monomeric form (Burz and Ackers, 1996
). Hence the identical nature of the spectra of the two repressors even at a concentration as high as 15 µM also suggests very little change in secondary structure following tetramer formation by the wild-type repressor. Clearly the secondary structure of S228N repressor is very similar to, if not identical with, that of the wild-type.
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Another way to measure destabilization of a protein by mutations is through measurement of denaturation. Figure 6 shows the effect of urea concentration on the F340/F350 ratio of S228N
-repressor. As the urea concentration increases from 0 to 10 M, there is a progressive shift of the emission maximum towards the red. The wild-type repressor shows two distinct transitions centered around 2 and 6.5 M urea (Deb et al., 1998
). In the case of S228N, the nature of urea denaturation is identical with that of the native repressor, i.e. the low urea transition occurs at 1.9 M urea and the high urea transition at 6.7 M urea. In the native S228N repressor, the F340/F350 ratio starts around 1.07 and levels off around 0.86 with an intermediate having an F340/F350 ratio of ~0.97, identical with that of the wild-type repressor under the same conditions. In 6.7 M urea, the emission maximum has shifted to 352.8 nm. In 9.6 M urea the emission maximum has shifted to 353.6 nm and the intensity at 340 nm has fallen to about 59.8% of that of the S228N repressor in the absence of urea. Hence, from the denaturation experiment, we conclude that the general integrity of the C-terminal domain structure is preserved in the mutant repressor.
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Discussion |
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If Gcoop is 2 to 3 kcal/mol and
Gint is ~8 kcal/mol (obtained from measurement of self-association of dimeroperator complexes), then the sum of
Gdis and
Gprox should be about 67 kcal/mol (negative sign indicating favored towards loop formation). Although we cannot measure
Gprox directly, we can estimate its magnitude. One possible way is to look at the effect of fusing two protein molecules on some properties that occur in trans. Such an approach was taken by Robinson and Sauer (1996a,b). In their study, and also other studies summarized in their papers, the effective concentration [Kd(bimolecular)/Kd(unimolecular) ratio] values are relatively small, ~3x103 M. Based on their results, we estimate that
Gprox is not very large. Hence loss of rotational and translational entropy upon binding to a piece of DNA does not contribute a great deal of free energy. This is not unanticipated since the only additional loss involved is loss of three degrees of rotational and three degrees of translational freedom (3RT
1.8 kcal/mol) (this is on the assumption that no preferential orientation occurs on the DNA). Hence
Gdis is likely to be ~8 kcal/mol with a lower limit of ~6 kcal/mol (i.e. when
Gprox is zero). Even the lower value of
Gdis indicates that a very significant DNA and/or protein distortion is present, consistent with CD and fluorescence studies (Roy et al., 1998
).
It is interesting that when the two operators were placed such that their separation is around 56 turns of DNA, the cooperative interaction occurred. If the proteinprotein interface in such a case is similar to the natural scenario, it would imply that the distortion to generate a loop of that size would be ~68 kcal/mol. This value is not inconsistent with the theoretical estimate (Hochschild and Ptashne, 1986).
The energetics of loop formation have a direct bearing on the mechanism of transcription activation and repression. In the differential contact model, the ability of the regulator to contact the appropriate intermediate or the transition state is crucial to the outcome, i.e. repression or activation. This ability to establish contact transiently is in turn dependent on favorable energies of loop formation during that time period. An understanding of the underlying energetics is therefore crucial for a mechanistic understanding of the process of activation and contact repression.
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Notes |
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Acknowledgments |
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References |
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Received January 7, 2000; revised June 21, 2000; accepted August 2, 2000.