The change of DNA structure by specific binding of the - Europe PMC

The EMBO Journal vol.3 no. 12 pp.2873 -2878, 1984

The change of DNA structure by specific binding of the cAMP receptor protein from rotation diffusion and dichroism measurements

D.Porschke', W.Hillen2 and M.Takahashi3 1Max-Planck-Institut fur Biophysikalische Chemie, 34 Gottingen, 2Technische Hochschule Darmstadt, Institut fur Organische Chemie, Petersenstrasse 22, 61 Darmstadt, FRG, and 3Laboratoire de Biophysique, Universite Paul Sabatier, 118, Route de Narbonne, 31062 Toulouse Cedex, France Communicated by T.Jovin

The structure of complexes formed between cAMP receptor protein (CRP) and various restriction fragments from the promoter region of the lactose operon has been analysed by measurements of electrodichroism. Binding of CRP to a 62-bp fragment containing the major site leads to an increase of the rotation time constant from 0.33 to 0.43 ps; addition of cAMP to the complex induces a decrease to 0.25 its. Similar data are obtained for a 80-bp fragment containing the operator site; however, in this case the decrease of the rotation time for the specific complex is only observed when the salt concentration is increased from 3 to 13 mM. A 203-bp fragment containing both sites showed a corresponding change after pre-incubation at 50 mM salt. The salt dependence of the rotation time for the specific complex formed with the 203-bp fragment also indicates that a compact structure is formed at 13 mM salt, whereas the structure is not as compact at 3 mM salt. A 98-bp fragment without specific CRP sites did not reveal changes corresponding to those observed for the specific fragments. The rotation time constants together with the dichroism amplitudes indicate that binding of CRP to speciflic sites in the presence of cAMP leads to the formation of compact structures, which are consistent with bending of DNA helices. The observed strong salt dependence of the structure is apparently due to electrostatic repulsion between adjoining helix segments. Key words: cAMP receptor/DNA/helix bending/rotation diffusion

Introduction The cAMP receptor protein (CRP, also known as catabolite gene activator protein CAP) regulates several genes in Escherichia coli (Zubay et al., 1970; De Crombrugghe et al., 1971; Epstein et al., 1975; Majors, 1975; De Crombrugghe and Pastan, 1978; Taniguchi et at., 1979; Ogden et al., 1980). When cAMP is present, CRP binds to specific DNA sites and promotes transcription. Various models have been proposed to explain the function of CRP at the molecular level. McKay and Steitz (1981) suggested from their X-ray analysis of CRP crystals that the protein binds to left-handed B-DNA. Salemme (1982) proposed that CRP binds to DNA in the form of a left-handed solenoid, which allows CRP to form contacts with the major grooves in successive loops of the solenoid. Recently it has been concluded from measurements of electrophoretic mobilities in polyacrylamide gels that DNA restriction fragments containing specific sites are bent upon binding of CRP (Kolb et al., 1983; Wu and Crothers, 1984). © IRL Press Limited, Oxford, England.

Since the CRP-DNA complex is of general interest as an example of protein-nucleic acid recognition and regulation, the structure of this complex should be analysed independently by methods without any possible influence of a gel matrix. The structure of DNA restriction fragments and their complexes with proteins may be analysed in dilute aqueous solution with high sensitivity by electro-optical measurements (Fredericq and Houssier, 1973; O'Konski, 1976; Hogan et al., 1978; Elias and Eden, 1981; Diekmann et al., 1982a,1982b). These measurements provide two independent pieces of information. The amplitude of the electrodichroism is a measure of the orientation of the base pairs relative to the long axis of the molecule. The time constants 7, observed after pulse termination, are directly related to the rotational diffusion coefficient and can be used to evaluate the molecular dimensions. Since the rotation time constants X are strongly dependent upon the length of rod-like molecules, the r values can be used for a very sensitive indication of changes in the apparent length. We have used this technique to investigate the binding of CRP to various DNA restriction fragments. Results Dichroism amplitudes Since the main goal of the present investigation is the analysis of the structure of a native protein-DNA complex, the conditions of the measurements had to be adapted to avoid any conformation change or dissociation reaction of the complex in the presence of the electric field. It is well known that complexes involving electrostatic interactions can be dissociated almost completely at high electric field strengths (Porschke, 1981; Diekmann and Porschke, 1982). Thus, our measurements had to be conducted at relatively low field strength. Due to the special construction of our pulse generator it is relatively difficult to generate pulses of low amplitude. For compensation we used a cell with a relatively large electrode distance of 13.5 mm. By this procedure we covered the range of electric field strengths from 1 to 30 kV/cm. Our measurements of the various restriction fragments in the absence of protein ligand provided electro-optical data very similar to those obtained previously for other fragments Diekmann et al., 1982a,1982b). Since the experiments were restricted to low electric field strengths, the limit dichroism at 'infinite' field strength could not be evaluated with sufficient accuracy. Some examples of the data are represented in Figure 1. Addition of CRP protein at a ratio of one protein molecule per DNA fragment leads to changes of the dichroism, which are dependent upon the DNA fragment and also upon the buffer concentration. As shown in Figure la the dichroism of the 62-bp fragment in the buffer T is clearly decreased on addition of CRP. A further and particularly strong decrease is observed on addition of 50 zM cAMP. The other fragments did not show similar strong effects upon ligand binding 2873

D.Porschke, W.Hillen and M.Takahashi

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Fig. 2. Change of light intensity AI as a function of time T after a field pulse of 11.2 kV/cm on a solution of the 80-bp fragment in buffer T. The experimental data and the fitted curve with a time constant of 0.51 its (convolution with detector time constant of 0.098 As) is shown in the upper part, while the differences between the experimental and the fitted curve are shown in the lower part. A small deviation indicating the appearance of a second relaxation process can already be recognised.

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Fig. 1. Reduced electric dichroism t of DNA fragments as a function of the electric field strength E without added protein (+), with one equivalent of CRP (0) and, in addition, with 50 /xM cAMP (*). (a) 62-bp fragment in buffer T; the amplitudes are corrected for a contamination as described in the text. (b) 80-bp fragment in buffer TIO. (c) 203-bp fragment; here the complex was formed with two equivalents of CRP in the presence of 50 /M cAMP and 50 mM NaCl + buffer T; the buffer was then exchanged for TlO + 50 /AM cAMP as described in the text.

under corresponding conditions. However, in the case of the 80-bp fragment the dichroism was more strongly affected by CRP + cAMP, when the ionic strength was increased by the addition of 10 mM NaCl (buffer TIO). An example is given in Figure lb for the 80-bp fragment. The dichroism of the 203-bp fragment was not changed very much by CRP + cAMP, even in the buffer T1O (Figure lc). The change of the dichroism observed upon addition of CRP + cAMP to the 98-bp fragment also remained relatively small. Rotational time constants The 80-bp fragment and its complex with CRP. Since the

time constants obtained from 'ON'-field curves (measured under electric field pulses) may be influenced by the electric field (Fredericq and Houssier, 1973; O'Konski, 1978; Diekmann et al., 1982b), we have evaluated only 'OFF'-field curves obtained at zero field strength. As described in the previous section we have also attempted to use relatively small field jumps to avoid field-induced reactions. The OFF-field curves obtained after small field jumps can usually be fitted 2874

+

0.50-\ 0.480.460.44 0

+

10

+

20

30 E 1kV/cm]

Fig. 3. Rotation relaxation time T for the 80-bp fragment in buffer T observed after termination of field pulses as a function of the electric field strength.

by single exponentials with sufficient accuracy (Figure 2). When the field strength is increased, a second exponential is observed very clearly for fragments with > 100 bp. Although in most cases a second exponential cannot be evaluated for the smaller fragments, the curves obtained for these fragments also show indications for the appearance of a second process already at relatively low field strengths. For example, the curves observed for the 80-bp fragment after field pulses up to 20 kV/cm can be fitted with good accuracy by single exponentials. However, the exponentials obtained in this range are not constant, but decrease with increasing field strength (Figure 3). When the field strength is increased beyond 20 kV/cm, the total change of transmission and the signal-to-noise ratio increase to values which are sufficient for the separation of two exponentials. The time constants T, obtained by fitting single exponentials over the whole range of field strengths E, show a linear correlation with E (Figure 3). Thus, a second process seems to be present already at low field strengths, but remains unresolved due to a low ampli-

Binding of the cAMP receptor protein

tude and/or low signal-to-noise ratio. This example demonstrates that fitting exponentials to experimental data is not always trivial. Usually the error sum of a fit for a given experimental curve can be decreased by allowing for another exponential component. Fitting an additional exponential component, however, often leads to a decrease in the accuracy of the numerical values for each of the components. This is a notoriously difficult problem, e.g., when one of the components is associated with small amplitude or when the time constants are very close to each other. Since a second exponential cannot be resolved at low field strength E, but appears to be reflected by the dependence of r values upon E, we have simply used the linear correlation of the experimental parameters for an extrapolation of the time constants to zero field strength. The resulting value ro may be regarded as representative of the overall rotational relaxation time at zero field strength. As shown in Figure 3, the time constants obtained from single jump experiments are associated with some experimental uncertainty, in particular at low electric field strengths. Since the linear correlation involves averaging, the T value extrapolated for zero field strength is relatively accurate and well reproducible. The r values measured for the CRP-DNA complexes have also been extrapolated to zero field strength by the same procedure. Addition of one equivalent CRP to the 80-bp fragment in the buffer T results in a slight decrease of the ro value from 0.57 Is for the free fragment to 0.56 Is for the protein-DNA complex. Although the dichroism curves are measured at very low concentrations (e.g., 0.38 ItM of the 80-bp fragment), we can be sure that the complex between CRP and the DNA is formed almost quantitatively. This may be concluded from measurements of CRP binding to DNA in the range of salt concentrations from 0.03 to 0.3 M (Baudras et al., 1983; Takahashi et al., 1983). Extrapolation of these data to the low salt concentration of the buffer T (ionic strength -3.3 mM) indicates that the equilibrium constant, even for unspecific binding, is > 1010 M- 1. Measurements of the rotation time constant for an equimolar mixture of CRP and 80-bp DNA in the presence of 50 ItM cAMP lead to a To value of 0.56 AsI. Thus binding of CRP to the 80-bp fragment in the buffer T does not lead to any major change in the apparent length of the DNA, both in the absence and the presence of cAMP. However, addition of NaCl to CRP + the 80-bp fragment in the presence of 50 IM cAMP leads to a clear decrease of the To value. At 2.5 mM Na + the To value is 0.27 As, at 5 mM Na + 7T is 0.20 ps and at OrM o is 0.13 As. For comparison we have also measured the time constants separately in the buffer TIO (i.e., buffer T + 10 mM NaCl). Under these conditions we found for the 80-bp fragment a To value of 0.49,s. Addition of one equivalent CRP led to a slight increase of To to 0.52 its, whereas addition of 50 IM cAMP induced a strong decrease of ro to 0.13 Its indicating the formation of a compact structure. These data show that formation of the compact structure requires both the presence of cAMP and a salt concentration of >3.3 mM. 62-bp fragment. Our sample of the 62-bp fragment was contaminated by some long DNA fragments. The contamination was clearly indicated by a dichroism amplitude associated with a slow rotation time constant of ro = 5.7 its. This dichroism amplitude was relatively large at low field strength, where long fragments, owing to their high dipole moments, are oriented more extensively than small fragments

Table I. Rotation relaxation times extrapolated to zero field strength in As at 200C

bp

Binding sites

62 80 98 203 80 98 203

1 2 Unspecific 1 + 2 2

Buffer

DNA

DNA+ CRP

DNA+cAMP+ CRP

T 0.33 0.43 0.25 T 0.56 0.56 0.57 T 0.88 0.91 0.88 T 4.5 4.7 2.4a 0.49 0.52 0.13 T10 0.81 TIO 0.81 0.84 Unspecific 1 + 2 3.7 1.48b T10 3.8 mM aThe sample was mixed in buffer T + 50 NaCl + 50 AM cAMP, then dialysed to buffer T + 50 uM cAMP. bSample after addition of 10 mM NaCl. The complexes were formed by addition of one equivalent CRP per DNA helix, except for the 203-bp fragment, where two CRP equivalents were added. The estimated accuracy of the time constants is i 27o in the range from 0.4 to I As. The accuracy is lower (about + 5%0) for the shorter time constants due to a lower signal to noise ratio and for the longer time constants due to the appearance of an additional fast process (cf. text)

(Diekmann et al., 1982a). However, at high field strengths the dichroism amplitude of the short fragment clearly exceeded that of the long fragment. Due to the rather low ro value of 0.33 As for the short fragment, the amplitudes could be separated at all field strengths. An analysis of the amplitudes indicated that our sample contained -7'7o of the long fragment.

Although the contamination impeded a complete and detailed analysis, it is possible to draw some conclusions from our measurements. Addition of one equivalent CRP to the sample resulted in an increase of the ro value to 0.43 Its. Subsequent addition of 50 tM cAMP induced a decrease of the ro value to 0.25 its. Thus, in analogy with the complex formed by the 80-bp fragment, cAMP leads to the formation of a compact conformation. In contrast to the '80-bp complex' the compact structure is formed with the 62-bp fragment already at the relatively low ionic strength of buffer T. 98-bp fragment. The fragment with 98 bp does not contain any specific binding site and was used for a control. Addition of one equivalent of CRP to this fragment in the buffer T increased its To value from 0.88 its to 0.91 As. When cAMP was added at a concentration of 50 AM the ro value decreased again to 0.88 its. Similar data were obtained in the buffer T10 (Table I). Thus CRP binding does not induce any major change in the structure of the 98-bp fragment. 203-bpfragment. The orientation curves for the 203-bp fragment required two exponentials for satisfactory fits already at low field strengths. Measurements in the buffer T provided an average slow rotation time constant T2 of 4.5 As. Addition of one equivalent CRP led to a slight increase Of r2 to 4.7 its and subsequent addition of 50 AM cAMP resulted in T2 values of 4.7 As. After further addition of a second equivalent of CRP we measured a T2 value of 4.7 its. Thus the measurements in buffer T did not reveal any significant change of the 203-bp helix dimensions after binding of CRP. Similar data were also obtained when CRP was added to the 203-bp fragment in the buffer T1O. Since the 203-bp fragment has two specific CRP sites and both of them separately exhibit relatively large changes of their dimension upon CRP binding, the results described above appear to contradict those obtained for the short fragments. If the absence of changes in the 203-bp fragment is 2875

D.Porschke, W.Hillen and M.Takahashi

203

Table II. Ionic strength dependence of rotation relaxation times extrapolated to zero field strength in 1ts at 20°C.

62

80i_

S21

Buffer

80 bp + CRP + cAMP

203 bp + 2CRP + cAMP

T T + 2.5 mM NaCI T + 5 mM NaCl T + 10 mM NaCl

0.56 0.27 0.20 0.13

2.4 1.85 1.48

The preparation of samples and accuracy are as described in Table L.

Fig. 4. Approximate relative sizes of DNA fragments and CRP (McKay and Steitz, 1981). The numbers denote the base pairs of the different fragments. The squares representing CRP are attached to the DNA helices at the centre of the specific binding sites. The major CRP site is denoted by I and the accessory site by 2 (cf. Schmitz, 1981). The contribution of CRP molecules to the overall dimensions would appear to be smaller, if the complexes were rotated around the long axis by 900.

due to trapping the CRP protein at unspecific sites, it should be possible to prepare the specific complex by incubation at high salt. Thus we mixed the 203-bp fragment with two equivalents of CRP in buffer T containing 50 mM NaCl and 50 AM cAMP. For dichroism measurements the mixture was then dialysed into buffer T containing 50 AM cAMP. The orientation curves obtained for this sample provided a slow time constant r2 of 2.4 As. After addition of 5 mM NaCl the r2 value decreased to 1.9 /s and in the presence of 10 mM NaCl a further decrease to 1.5 /ts was observed. These results demonstrate that there are two factors which inhibit the formation of specific and compact complexes. The reduction of the r2 value after pre-incubation at high salt shows that CRP is trapped at unspecific sites when added to long fragments at low salt. The dependence of the rotation time upon the salt concentration indicates that electrostatic repulsion between helix phosphates inhibits the formation of compact structure at low salt. The fast time constant observed for the 203-bp fragment is -0.5 its. This value remains almost unaffected when CRP + cAMP is added.

a

Discussion Rotational relaxation time constants are known to be strongly dependent upon molecular dimensions. In the case of DNA helices the rotation time is mainly determined by the helix length. The chain length dependence of observed in the range up to -300 bp, can be described with satisfactory accuracy by a weakly bending rod model (Kuhn et al., 1953; Hearst, 1963; Elias and Eden, 1981; Diekmann et al., 1982b). According to this model in the form developed by Hearst (1963) the reciprocal time constant is given by: r

T,

I Xr

6kT

13 1n(L/b)- 7+4(b/a)+XL[2.25 ln(L/b) -6.66+2 (b/a)]J -

-

-

prL3

where k is the Boltzmann constant, Tis absolute temperature, -1 is solvent viscosity, L is contour length of the rod, b is distance between frictional elements, a is Stokes diameter of each element and 1/X is statistical length corresponding to twice the persistence length. The rotation time is usually assumed to be independent of the buffer and its concentration. Our present measurements (Table I) together with other data show, however, that the buffer may have a relatively strong influence on the rotation time (in preparation). For the purpose of the present investi2876

gation it is not necessary to analyse this effect in detail. We simply use the time constants determined for the free fragments in the respective buffers as reference values. When a CRP molecule is bound to a DNA fragment, the rotation time constant of the complex should be larger than that of the free fragment, provided that the structure of the DNA helix remains unchanged. Binding of CRP to our DNA fragments actually leads to an increase of the r values in almost all cases (Table I) provided that the solutions do not contain cAMP. This increase is relatively large for the shortest fragment containing 62 bp and very small for the longest fragment with 203 bp. The dependence of the increase upon the helix length is qualitatively consistent with the relative size of the molecules (Figure 4). As a first approximation the rotation times of the complexes may be estimated from the harmonic means of the diffusion coefficients for the individual molecules (cf. Austin et al., 1983) corresponding to additivity of the time constants. From the size of the CRP dimer (McKay and Steitz, 1981) it may be estimated that its rotational correlation time is in the range 20-40 ns. Most of the experimental values for CRP-DNA complexes are consistent with the value expected from the simple harmonic approximation. Deviations observed in some cases may be due to slight changes of the DNA structure and/or flexibility. Since these deviations remain relatively small, however, it may be concluded that binding of CRP does not lead to any major change of helix structure under our experiment conditions. Compared with the relatively small change of the rotation time constants induced by the binding of CRP, the changes induced by the addition of cAMP to the CRP complexes with specific DNA are quite dramatic. The strong decrease of the r values induced by cAMP clearly demonstrate a conformation change of the complexes resulting in a large reduction of the effective hydrodynamic dimensions. This effect may be explained by various molecular models. It is conceivable, for example, that the helix is turned to a more flexible state at the CRP binding site. The decrease of the rotation time may also be explained by the formation of a DNA cruciform. Finally it is possible that the DNA rod is bent by CRP in the presence of cAMP. All these models result in a reduction of the effective hydrodynamic dimensions and thus a reduction of the overall rotation time constant. The main elements of the different models may also be combined with each other. For example, it is possible that a cruciform does not only decrease the apparent length, but may also introduce flexibility at the cruciform site. An analysis of the DNA sequences shows that the possible cruciforms would reduce the effective length by an amount which would just be sufficient to explain the strong reduction of the rotation time constants. Thus, it appears to be impossible to distinguish between these models by measurements of rotation time constants alone.

Binding of the cAMP receptor protein

Some additional information on the structure of the complex is available from the dichroism amplitudes. As shown in Figure 1 the change of the dichroism amplitude upon CRP binding to the specific DNA fragments remains relatively small when cAMP is not present. A corresponding result is obtained for the CRP complex with the unspecific 98-bp fragment (data not shown). These observations are consistent with the rotation time constants and demonstrate that binding of CRP to DNA in the absence of cAMP does not lead to any major change of the DNA structure. These data also show that the dipole induced at the DNA helix is not strongly affected by the presence of the protein. Compared with the relatively small changes of the dichroism amplitudes induced by CRP binding, the changes induced by addition of cAMP to the CRP complexes with the 62-bp and 80-bp fragments are remarkably strong and indicate (in agreement with the rotation time constants) a large change of the conformation. Although all the models mentioned above are consistent with a decrease of the dichroism, it seems that the model involving an increased flexibility does not explain the observed effects without problem. It is likely that the electric field would tend to align the segments connected by a flexible joint and thus lead to dichroism amplitudes not too far from those found for the helix without a flexible joint. The effects observed for the 203-bp fragment are particularly interesting, since the dichroism is reduced by a small degree only, whereas the rotation time constant is reduced considerably. This observation may be explained by sharp bending such that the helix stems are arranged almost parallel to each other. The strong electrostatic repulsion indicated by the dependence of the rotation time constants upon the salt concentration (Table II), is consistent with sharp bending of the helix and a close contact of the helix segments in the compact state. However, if both specific sites, which are present in the 203-bp fragment, were to be sharply bent, the rotation time constant would still be much smaller than found in our experiments. Since it may be expected that the electrostatic repulsion for a sharply bent 203-bp fragment is higher than for a corresponding 80-bp fragment, it is possible that the rotation time constant of the 203-bp + CRP + cAMP complex is further reduced at higher salt concentrations. More detailed conclusions on the structure of the CRP complex may be obtained by a comparison of our experimental data with hydrodynamic model calculations. Some impression on the magnitude of the conformation changes induced by CRP + cAMP may be obtained by simple estimates on the basis of the weakly bending rod model. If we assume, for example, that the 80-bp fragment is sharply bent at the centre of the CRP binding site, we arrive at a rotation time constant of -0.13 As. This value is identical with the experimental value, although the contribution of the protein molecule itself has not been considered. Obviously the model used for comparison is over-simplified. Nevertheless the comparison indicates that the DNA is converted to a very compact form on binding of CRP + cAMP. This may also be concluded from a comparison of our data with the results of model calculations by Mellado and Garcia de la Torre (1982) on various rigid bent rods. Our results obtained from electro-optical measurements in dilute aqueous solution are consistent with recent interpretations of the anomalous mobility of specific CRP-DNA complexes in polyacrylamide gels (Kolb et al., 1983; Wu and Crothers, 1984; cf. also Marini et al., 1982). Since the mobility of macromolecular complexes in gels may be influenced

by many different factors, evidence obtained from gels may not be regarded as conclusive. However, the combination of results obtained by independent methods should be convincing. It may be safely concluded that CRP + cAMP binding leads to an unusual conformation of specific DNA with a clear reduction of the effective hydrodynamic dimensions. Since the relatively low mobility of the complexes observed by gel electrophoresis can hardly be explained by an increase of DNA flexibility, we may exclude the 'flexible joint' model discussed above. Furthermore, the formation of cruciforms seems to be unlikely from binding experiments to closed circular DNA (Kolb and Buc, 1982). Thus DNA bending remains as the most likely explanation for the unusual properties of CRP + cAMP complexes with specific DNA. Recently it has been shown by X-ray crystallography that binding of EcoRI endonuclease to its cognate site on a dodecamer helix results in 'kinks' associated with some bending of the helix (Frederick et al., 1984). Thus, kinks and bending of DNA helices may be of general importance for specific proteinnucleic acid interactions. Materials and methods CRP was purified to homogeneity from an over-producing strain harbouring the plasmid pBScrp2 (Cossart and Gicquel-Sanzey, 1982) using the procedure described previously (Takahashi et al., 1980). The DNA fragments were prepared from pRW574 essentially as described by Hillen et al. (1981). The final purification was done by chromatography on RPC-5 (Hillen and Wells, 1983). Details of the purity and biological activity of the DNAs were as described elsewhere (Hillen and Wells, 1980). The electric dichroism was measured with an improved version of the apparatus described by Diekmann et al. (1982a). To avoid photoreactions, illumination of the samples was restricted by an automatic shutter to a few seconds for each field jump experiment. This time interval was also used to determine the 'total' light intensity (I) before the electric field jump; the value of I was stored by a sample and hold circuit. Both the light intensity and the electric field were recorded as a function of time by a two-channel transient digitizer 'tektronix 7612D'. The data were transferred to a LSI 11/23 (digital equipment), which was used to evaluate amplitudes of both light intensity and electric field strength with the aid of graphic routines. For the analysis of time constants the data were transmitted to the Univac 1108 of the Gesellschaft fur wissenschaftliche Datenverarbeitung, Gottingen; exponentials were fitted by procedures described previously (Diekmann et al., 1982b). Our standard buffer (T) contained 5 mM tris-hydroxymethylaminomethane, 0.1 mM EDTA and 0.1 mM 1,4-dithioerythritol pH 8.0. For some measurements the ionic strength was increased by the addition of 10 mM NaCl (buffer TIO).

Acknowledgements The expert technical assistance of Jorg Ronnenberg and the CRP preparation of Dr B.Blazy are gratefully acknowledged. We are indebted to Dr T.M.Jovin for his comments on the manuscript. M.T. thanks Professor A.Baudras for his encouragement.

References Austin,R.H., Karohl,J. and Jovin,T.M. (1983) Biochemistry (Wash.), 22, 3082-3090. Baudras,A., Blazy,B. and Takahashi,M. (1983) Biochimie, 65, 437-440. Cossart,P. and Gicquel-Sanzey,B. (1982) Nucleic Acids Res., 10, 1363-1378. De Crombrugghe,B., Chen,B., Anderson,W., Nissley,P., Gottesmann,N. and Pastan,1. (1971) Nature New Biol., 231, 139-142. De Crombrugghe,B. and Pastan,I. (1978) in Miller,J.H. and Reznikoff,W.S. (eds.), The Operon, Cold Spring Harbor Laboratory Press, NY, pp. 303324. Diekmann,S., Hillen,W., Jung,M., Wells,R.D. and Porschke,D. (1982a) Biophys. Chem., 15, 157-167. Diekmann,S., Hillen,W., Morgeneyer,B., Wells,R.D. and Porschke,D. (1982b) Biophys. Chem., 15, 263-270. Diekmann,S. and Porschke,D. (1982) Biophys. Chem., 16, 261-267.

Elias,J.G. and Eden,D. (1981) Macromolecules, 14, 410-419. Epstein,W., Rothmann-Denes,L.B. and Hesse,J. (1975) Proc. Nati. Acad. Sci. USA, 72, 2300-2304. Frederick,C.A., Grable,J., Melia,M., Samudzi,C., Jen-Jacobson,L., Wang,

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D.Porschke, W.Hiilen and M.Takahashi B.-C., Greene,P., Boyer,H.W. and Rosenberg,J.M. (1984) Nature, 309, 327-331. Fredericq,E. and Houssier,C. (1973) Electric Dichroism and Electric Birefringence, published by Clarendon Press, Oxford. Hearst,J.E. (1963) J. Chem. Phys., 38, 1062-1065. Hillen,W. and Wells,R.D. (1980) Nucleic Acids Res., 8, 5427-5444. Hillen,W. and Wells,R.D. (1983) Techniques in Life Sciences, B5 Nucleic Acid Biochemistry, B512, 1-17. Hillen,W., Klein,R.D. and Wells,R.D. (1981) Biochemistry (Wash.), 20, 3748-3756. Hogan,M., Dattagupta,N. and Crothers,D.M. (1978) Proc. Natl. Acad. Sci. USA, 75, 195-199. Kolb,A. and Buc,H. (1982) Nucleic Acids Res., 10, 473-485. Kolb,A., Spassky,A., Blazy,B. and Buc,H. (1983) Nucleic Acids Res., 11, 7833-7852. Kuhn,H., Kuhn,W. and Silberberg,A. (1953) J. Polym. Sci., 14, 193-208. Majors,J. (1975) Nature, 256, 672-674. Marini,J.C., Levene,S.D., Crothers,D.M. and Englund,P.T. (1982) Proc. Natl. Acad. Sci. USA, 79, 7664-7668. McKay,D.B. and Steitz,T.A. (1981) Nature, 290, 744-749. Mellado,P. and Garcia de la Torre,J. (1982) Biopolymers, 21, 1857-1871. Ogden,S., Haggerty,D., Stoner,C.M., Koladrubetz,D. and Schleif,R. (1980) Proc. Natl. Acad. Sci. USA, 77, 3346-3350. O'Konski,C.T. (1976) Molecular Electro-optics, part 1, published by Dekker, NY. O'Konski,C.T. (1978) Molecular Electro-optics, part 2, published by Dekker, NY. Porschke,D. (1981) in Krause,S. (ed.), Molecular Electro-Optics, Plenum, NY, pp. 269-284. Salemme,F.R. (1982) Proc. NatI. Acad. Sci. USA, 79, 5263-5267. Schmitz,A. (1981) Nucleic Acids Res., 9, 277-292. Taniguchi,T., O'Neill,M. and De Crombrugghe,B. (1979) Proc. Natl. Acad. Sci. USA, 76, 5090-5094. Takahashi,M., Blazy,B. and Baudras,A. (1980) Biochemistry (Wash.), 19, 5124-5130. Takahashi,M., Blazy,B., Baudras,A. and Hillen,W. (1983) J. Mol. Biol., 167, 895-899. Wu,H.M. and Crothers,D.M. (1984) Nature, 308, 509-513. Zubay,G., Schwartz,D. and Beckwith,J. (1970) Proc. NatI. Acad. Sci. USA, 66, 104-110.

Received on 10 August 1984

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