on the nature of dna promoter conformations : the effects of glycerol and dimethylsulphoxide

Eur. J. Biochem. 47,435-441 (1974)

On the Nature of DNA Promoter Conformations The Effects of Glycerol and Dimethylsulphoxide

Andrew TRAVERS

Medical Research Council, Laboratory of Molecular Biology, Cambridge

(Received April 17/June 13, 1974)

Low concentrations (up to 20 "/, v/v) of glycerol and dimethylsulphoxide stimulate RNA synthesis in vitro, the extent of stimulation being dependent on the nature of the DNA template. This stimulation results mainly from an increase in initiation. The target of these compounds is the DNA template rather than RNA polymerase. Ribosomal RNA synthesis is affected in a temperature- dependent manner, 20 % glycerol lowering the transition temperature between the open and closed forms of the promoter by 4- 5 "C. Thus in a manner analogous to its effect on gal mRNA synthesis in vitro glycerol partially releases rRNA synthesis from the constraints essential for control.

The first step in the synthesis of an RNA molecule is the interaction of RNA polymerase with a particular site on the DNA template. It is presumably this interaction which determines the specificity of transcription. Consequently we might expect that alterations in the structure of either the polymerase or the template could result in changes in specificity. Among the factors which can directly affect the ability of a given DNA molecule to act as a template are the temperature [l - 41, the superhelicity of a closed DNA duplex [5 - 71 and the presence of ethylene glycol [8]. Under certain conditions temperature operationally defines two states of a specific promoter, that for ribosomal RNA synthesis; above 36 OC the promoter is open, below 35 "C it is closed [4]. The transition temperature between these two states can be altered by molecules which interact directly with the template. It is, for example, raised by a DNA-binding protein, H I protein [9].

In this paper I show that two rather dissimilar compounds, glycerol and dimethylsulphoxide, have very similar effects on transcription in vitro. At low concentrations (up to 20 "/, v/v) both stimulate total RNA synthesis, the extent of stimulation depending on the type of DNA template. This stimulation results mainly from an increase in initiation and is a con- sequence of the action of the compounds on the DNA template. Ribosomal RNA synthesis is affected such that the transition temperature between the two states of the promoter is lowered by 4- 5 "C in the presence

Enzymt,. Nucleosidetriphosphate : RNA nucleotidyltrans- ferase or RNA polyiiierase (EC 2.7.7.6).

of 20% (v/v) glycerol. Since the characteristics of the control of ribosomal RNA synthesis in vivo are paralleled most closely in vitro from the closed state to rhe promoter [lo], the effect of glycerol is thus partially to release rRNA synthesis in vitro from the constraints essential for control. A striking analogy to this effect exists in the lactose system where the requirement for cup protein for gal mRNA synthesis in vitro is abolished by glycerol [ll].

MATERIALS AND METHODS

Prepuration of R N A Polymerase and E . coli D N A

RNA polymerase holoenzyme was prepared from E. coli CA 285 by the method of Burgess and Tra- vers [12]. The enzyme preparation was > 95 % pure as judged by electrophoresis in 7.5 % polyacrylamide gels in 0.1 % sodium dodecylsulphate. The enzyme was stored at 4 "C in 20 % glycerol, 0.01 M Tris-HC1 pH 7.9 at 20"C, 0.01 M MgCl,, 0.5 M KCl, and 0.1 mM dithiothreitol at a concentration of 2.2 mg/ml.

E. coli DNA was prepared from E. coli MRE 600 by a modification of the method of Marniur[13]. 20 g bacteria were suspended in 100 ml 0.1 M NaC1, 0.05 M EDTA (Na salt) pH 7,0.05 M Tris-HC1 pH 8.5. Sodium dodecylsulphate was added to a final concentration of 0.1 :( and the resulting lysate gently mixed. Self-digested pronase [14] was then added to 200 pg/ml and the lysate was incubated at 37°C for 18 h. This digest was gently extracted twice with an equal volume of water-saturated phenol and then dialysed against

Eur. J. Biochem. 47 (1974)

436 DNA Promoter Conformations

three changes of 0.1 M KCl, 0.1 mM EDTA, 0.01 M Tris-HC1 pH 7.9. Pancreatic ribonuclease, previously preincubated at 80 "C for 30 min, was added to 10 pg/ ml and the DNA preparation was incubated for 3 h at 37 "C. Pronase was then added to 20 pg/ml and the incubation continued for a further 4 h at 37°C. The DNA preparation was again extracted with phenol, dialysed, and then underwent a further cycle of treat- ment with ribonuclease and pronase. The DNA was finally purified by phenol extraction and dialysis. This method of preparation yields DNA at a concentration of 0.5- 1 mg/ml. When such a DNA preparation is analysed by electrophoresis on 10 % polyacrylamide gels in 0.1 % sodium dodecylsulphate no detectable individual polypeptide chains are visualised on staining with Coomassie brilliant blue.

R N A Polymerase Assay

Assay mixtures (final volume 100 pI) contained 0.05 M Tris-HC1 pH 7.9, 0.1 mM EDTA, 6 mM 2-mercaptoethanol, 10 mM MgC12, 250 pM each of ATP, CTP and GTP, 25 pM [14C]UTP (specific activity 43 Ci/mol) and 50 pg/ml DNA. Except where otherwise stated the reaction mixtures were incubated for 5 min at 33 "C and RNA synthesis was started by the addition of RNA polymerase to 20 pg/ml. The glycerol concentration due to addition with polymerase was 0.2%. The reaction was further incubated for 10 min at 33 "C and terminated by the addition 3 ml 5 % trichloroacetic acid, 0.01 M pyrophosphate. The precipitate was collected on Millipore HAWP 25-mm discs, which were then rinsed three times with 5 % trichloroacetic acid. Discs were finally counted for radioactivity in a toluene-based scintillator using a Nuclear Chicago 720 scintillation counter.

Preparation of R N A for Hybridisation Analysis

The reaction mixtures for RNA synthesis in vitro contained in a final volume of 0.2 ml: 0.04 M Tris- HC1 pH 7.9, 0.1 M KCl, 0.01 M MgC12, 6 mM 2-mercaptoethanol, 0.1 mM EDTA, 0.25 mM each of ATP, CTP and GTP, and 3 pM [32P]UTP (specific activity 19000 Cilmol from New England Nuclear), E. coli DNA 100 pg/ml and RNA polymerase 40 pg/ ml. Before the addition of RNA polymerase the reaction mixture was incubated for 10 min at the indicated temperature. RNA synthesis was then started by the addition of enzyme and allowed to proceed for 15 min at the same temperature. The reaction was terminated by the addition of pancreatic DNase to a final concentration of 50 pg/ml. The incubation was continued for a further 10 min after which the reaction mixture was diluted with 0.2 ml 4 x standard saline citrate.

The resulting solution was shaken with 0.4 ml water- saturated phenol. The aqueous phase from this extraction was used directly as a source of RNA for hybridisation. The amount of total RNA synthesis was determined by trichloroacetic acid precipitation of a duplicate 20-p1 aliquot of the aqueous phase.

Determination of rRNA in the in vitro Transcript The amount of rRNA in labelled E. coli RNA was

determined by hybridisation in triplicate of 50-p1 aliquots of RNA to denatured E.coli DNA in the presence and absence of 6 pg/ml unlabelled rRNA [4]. The hybridisation efficiency in vivo of 32P-labelled rRNA to E. coli DNA varied between 16 % and 25 % in the experiments described .

Assay of Rifampicin-Resistant Complexes of R N A Polymerase and T4 DNA

Rifampicin-resistant complexes were assayed es- sentially as described by Bautz et al. [3]. RNA polymerase holoenzyme (40 pg/ml) and DNA (160 pg/ml) were preincubated in a standard reaction mixture lacking the four nucleoside triphosphates for 10 rnin at the indicated temperature. The nucleoside triphosphates, including ['4C]UTP (specific activity 43 Ci/ mol), were then added together with rifampicin (final concentration 10 pg/ml). The reaction mixture was immediately shifted to a temperature of 38°C and RNA synthesis allowed to proceed for 10 rnin at that temperature.

RESULTS

Effect of Glycerol and Dimethylsulphoxide on Overall Transcription

Glycerol is a common component of storage buffers for RNA polymerase [12,15,16]. Consequently it is of interest to determine whether this compound can itself influence transcription. With E. coli DNA as template addition of glycerol to an RNA polymerase reaction mixture increases total RNA synthesis up to a glycerol concentration of 20% (v/v) (Fig. 1). The response over this range is approximately linear and results in up to a 3-fold increase in total RNA synthesis. The synthesis of RNA in the presence and absence of glycerol is linear for at least 60 min in this system (Travers, unpublished observations). Thus the observed increase in total synthesis represents an increase in the rate of RNA synthesis. At glycerol concentrations greater than 20 % (v/v) transcription is progressively inhibited. With T4 DNA as template a different response to glycerol is observed, RNA synthesis being only slightly stimulated (up to 20%) at


A. Travers

150

125

- - E

0 5 loo E

- 0

Q - n .- g 75 E u c ._ a z =I 50

25

C

\

\ T 4 DNA

10 20 30 Glycerol ( % , v i v )

Fig. 1. Effect of glycerol on transcription in vitro of T4 and E.coli DNA. Reaction conditions were as described in Materials and Methods

10 % (v/v) glycerol. Increasing the glycerol concentration further then results in a strong inhibition of RNA synthesis. Thus the extent and character of the effect of glycerol on transcription is strongly dependent on the nature of the DNA template.

Since the effect of glycerol on transcription was template-dependent, dimethylsulphoxide (a compound known to affect DNA structure) was also tested for its effect on transcription. The results (Fig. 2) show that dimethylsulphoxide influences the transcription of E. coli DNA and T4 DNA in a very similar manner to glycerol, maximum stimulation of RNA synthesis on both templates being observed at the same concentration (v/v) as glycerol. Thus on a molar basis glycerol and dimethylsulphoxide appear to be equally effective. The only difference between the compounds was a reproducibly slightly greater stimulation of total RNA synthesis by dimethylsulphoxide than by glycerol.

In addition to glycerol and dimethylsulphoxide, the effects of sucrose, formamide and isopropanol on transcription were also tested. While sucrose up to 30 % (w/v) had no significant effect on RNA synthesis from either E. coli DNA or T4 DNA, both isopropanol and formamide strongly inhibited transcription over

150

125 - - E - 0

g loo

B g 75

. - a - s " c .- a I 3

50

25

0

437

b

10 20 30 I I I

MezSO ( % , v i v )

Fig. 2. Effect of dimethylsulphoxide (Me2SO) on transcription in vitro of T4 and E. coli DNA. Reaction conditions were as described in Materials and Methods

the same concentration range (data not shown). These compounds were not investigated further.

Effect of Glycerol and Dimethylsulphoxide on Initiation and Elongation of RNA Chains

What is the mechanism of the stimulation of RNA synthesis by glycerol and dimethylsulphoxide ? To test whether these compounds increased the initiation or elongation of RNA chains, or indeed affected both these processes, RNA polymerase and the DNA template were first incubated in the absence of the ribonucleoside triphosphates at 34 "C. Under these conditions some polymerase molecules form a complex with a promoter site on the DNA. Such polymerase molecules are relatively resistant to the antibiotic, rifampicin [17 - 191. Consequently by adding the tested compound before or immediately after the simulta- neous addition of rifampicin and nucleoside triphosphates we can test whether the compound increases the formation of rifampicin-resistant complexes, i.e. increases initiation, or whether it increases the rate of elongation of RNA chains initiated from pre- existing rifampicin-resistant complexes. Table 1 shows



Table 1 . EJfect of preincubation of R N A polymerase and DNA with glycerol and dime fhylsulphoxide The reaction conditions were as described in the final section of Materials and Methods except that in Expt 2 [E-~~PJUTP (specific activity 1100 Ci/mol) was used in place of [14C]UTP. EG = RNA polymerase, Me,SO = dimethylsulphoxide, rif = rifampicin

Expt Preincubation Additions RNA synthesis

1

2

EG + T4 DNA Ea + T4 DNA

EG + T4 DNA + 10% Me,SO

EG + E. coli DNA Ea + E. coli DNA

- ~-~

EG + E.coliDNA + 20 % glycerol

counts/min rif + NTPs 1535 rif + NTPs + 10% MezSO 2077

rif + NTPs 3001

rif + NTPs 1335 rif + NTPs + 20 % glycerol 181 3

rif + NTPs 5111

-~ ~~ ~

that with E. coli DNA as template addition of 20'7, glycerol prior to rifampicin increases the observed RNA synthesis by about 400%. In contrast addition of 20 % glycerol immediately after rifampicin increases RNA synthesis by only 30%. Thus the major effect of glycerol is to increase the formation of rifampicin- resistant complexes and so to increase initiation. Nevertheless the data suggest that glycerol may also have a small effect on chain elongation. In a similar experiment using a T4 DNA template dimethylsulphoxide stimulated RNA synthesis by 100% and 30% when added prior to and immediately after rifampicin respectively (Table 1). In this case also the major effect of the added compound was on initiation but again a significant effect on chain elongation was noted.

Target of Glycerol and Dimethylsulphoxide

Glycerol and dimethylsulphoxide could affect transcription by altering the structure of either the DNA template or RNA polymerase. To determine the target of these compounds E. coli DNA and RNA polymerase were preincubated separately in their presence and absence. The missing component of the reaction mixture was then added and RNA synthesis resulting over a short period was then measured. Table 2 shows that both glycerol and dimethylsulphoxide significantly increase total synthesis only when preincubated with the DNA template. The slight stimulation of synthesis observed when glycerol or dimethylsulphoxide is preincubated with RNA polymerase is statis- tically not significant. Thus the target of glycerol and dimethylsulphoxide must be the DNA template

Table 2. effect ofpreincubation of RNA polymerase and E. coli D N A separately with glycerol and climetlx,vlsu(pltoxide Either E. coli DNA or RNA polymerase was preincubated with and without the added compounds in a reaction mixture lacking nucleoside triphosphates for 3 min at 33 "C. The nucleoside triphosphates including ['4C]UTP (specific activity 43 Ci/mol) were then added as indicated together with either DNA or RNA polymerase, which had been separately preincubated for 3 min at 33 "C. RNA synthesis was then allowed to proceed for 2 min at 33 "C. Me2S0 = dimethylsulphoxide, EG = RNA polymerase

Expt Preincubation Additions RNA synthesis

counts/min 1

-_ 2

E. coli DNA E. coli DNA + 20% MezSO EG EG $. 20% Me,SO

E. coli DNA E.coliDNA + 20 glycerol EG Em + 20 ?( glycerol

- _ _ ~

EG

EG E. coli DNA E. coli DNA

EG

En E. coli DNA E. coli DNA

~

296

1410 246 422

332

1043 20 1 318

and not RNA polymerase. Since the RNA synthesis measured in this experiment requires the participation both of RNA polymerase and DNA, it could be argued that glycerol or diinethylsulphoxide might act on the polymerase. DNA complex rather than on either component individually. If this were the case the order of addition of the components should not affect the stimulation observed. In fact the order of addition appears to be of critical importance and consequently the stimulation of RNA synthesis by glycerol or dimethylsulphoxide could only influence the complex by affecting the structure of DNA outside that immediately protected by the polymerase.

It is important in experiments of this type that the DNA, which is added to a preincubated mixture containing polymerase, also be preincubated at the same temperature. Addition of DNA preincubated at 4°C results in significantly less synthesis than when RNA polymerase is added to 33°C preincubated DNA.

Effect of Glycerol on Ribosomal RNA Synthesis

To investigate further the mechanism of action of glycerol its effect on the transcription of a specific RNA species, E.coli ribosomal RNA, was investigated. liz vitro the rate of synthesis of this RNA species is strongly temperature-dependent, the absolute and proportional rate at 37 "C being five times that at 34°C. Outside this temperature range the rate of increase of rRNA synthesis corresponds merely to a doubling per 10 "C rise [4]. When increasing amounts


A. Travers

4'0,

"0 5 10 15 20 25 30 35 Glycerol (o/o,v/v)

Fig. 3. Eflect of glycerol on r R N A transcription at 33 "C. RNA was prepared and rRNA was assayed as described in Materials and Methods. The input in the hybridisation assay for the RNA samples synthesised in the absence of added glycerol was 24300 counts/min (0) and 26700 counts/min (A). The input for other samples relative to this sample was proportional to total RNA synthesis which followed the curve shown in Fig. 1. (0, A) Represent data from separate experiments

of glycerol were added at 33 "C, when the rate of rRNA synthesis is normally low, the proportion of rRNA in the transcript was significantly increased (Fig. 3). In the absence of added glycerol rRNA comprised < 2 % of the transcript, whereas at 5 , 10 and 20% (v/v) glycerol rRNA comprised about 7, 11 and 15% of the transcript, respectively. The glycerol-induced increase in total RNA synthesis in this experiment was substantially the same as that observed in previous experiments (Fig. 1) and the increase in the absolute rate of rRNA synthesis was at least 20-fold at 20% (v/v) glycerol.

It should be noted that low concentrations of glycerol. for example up to 5% (v/v), significantly increase the proportional rate of rRNA synthesis. Consequently it is not possible to directly compare the data shown here with other studies on rRNA synthesis in vitro.

The effect of glycerol on rRNA synthesis over an extended temperature range is shown in Fig.4; 5 % (v/v) glycerol stimulates rRNA synthesis most mark- edly at 33°C but has no effect at 25°C or 29°C. At 38 "C a slight and reproducible stimulation of rRNA synthesis is observed. This pattern is emphasised by 20% (viv) glycerol. Again no significant alteration in rRNA synthesis is observed at 25°C or 28°C even though total RNA synthesis is strongly stiinulated.

439

. Glycerol

3000 - (olo8 v l v )

Temperature ( "C) Fig. 4. Effect of glycerol on r R N A irunscripiion us a function of temperature. Total E. coli RNA was prepared and rRNA was assayed as described in Materials and Methods. The data in Fig. 4 for 33 "C are transposed to this figure for reference. (A) Points for 20 % dirnethylsulphoxide

However, at 33 "C and 38 "C rRNA is synthesised at a constant high rate. This rate is about three times the maximum rate observed in the absence of glycerol.

Thus glycerol appears to have two major effects on rRNA synthesis. First the transition temperature, t , between the open and closed forms of the promoter, which define the rates of synthesis [4], is lowered. In the absence of added glycerol t was approximately 36 "C. In 5 % and 20 "/, (v/v) glycerol t was calculated to be about 32 "C and 30 - 31 "C, respectively. Second- ly, glycerol increases the maximum rate in vitro of expression of the rRNA genes. Since glycerol acts by stimulating initiation and its overall target is the DNA template, the effect of glycerol must arise from a change in the environment of the promoter. Thus these observations confirm the inference that the conformation of a promoter can determine the rate of RNA synthesis [4].

The pattern of rRNA synthesis in the presence of 20 % (v/v) dimethylsulphoxide was very similar to that observed in the presence of 20% (v/v) glycerol (Fig. 4). Thus dimethylsulphoxide also lowers the transition temperature between the two forms of the promoter.

Effect of Glycerol on T4 RNA Synthesis

To test whether the temperature dependence of the glycerol effect was a more general phenomenon the effect of glycerol on the formation of stable polymerase . DNA complexes at T4 DNA promoters was



I 1 I 10 20 30

Temperature (“C)

Fig.5. Effect of 10% glycerol on the formation of rifarnpicin- resistant polymerase . promoter complexes on T4 DNA. Reac- tion conditions were as described in Materials and Methods. (0) No added glycerol; (0) with 10 % added glycerol

determined. Such complexes are relatively resistant to the antibiotic rifampicin [18,19] and so can be assayed by preincubating the polymerase and template and then adding rifampicin together with the nucleotide triphosphates. Fig. 5 shows that 10 % glycerol lowered the temperature required for 50 % complex formation by about 16 “C. This depression is substantially greater than that observed for rRNA synthesis. However, the experiments are not strictly comparable for in this experiment using T4 DNA, complex formation and dissociation attains equilibrium [18, 191 whereas in the case of rRNA synthesis dissociation of the complex must compete with initiation of RNA synthesis. Nevertheless it is clear that the effect of glycerol is qualitatively the same for ribosomal and T4 RNA synthesis.

The transition temperature, approximately 26 “C, for the formation of the stable complex in the absence of glycerol is substantially higher than that observed by Zillig et al. [20] for the same template. This is to be expected [21], since the concentration of mono- valent cations used in the current experiment is twice that used in the other published experiments.

DISCUSSION The experiments described here show that at low

concentrations both glycerol and dimethylsulphoxide can stimulate transcription in vitro by increasing the

initiation of RNA chains. The target of these compounds is the DNA template rather than RNA polymerase. The effects of these compounds on a molear basis are quantitatively very similar and thus it seems likely that their modes of action are the same. But in what way could they alter the structure and environment of the DNA template? The most direct evidence comes from experiments on the effect of glycerol on rRNA synthesis. In this system a major effect is the reduction in the presence of glycerol of the transition temperature between the open and closed forms of the DNA in the promoter regions for rRNA synthesis. Thus, much of the stimulation by glycerol of the absolute and proportional rates of rRNA synthesis occurs over a limited temperature range and is the results of the conversion of an operationally closed promoter to an open promoter. The effect of glycerol is, however, not restricted to rRNA synthesis. The compound also lowers the transition temperature for the formation of “rifampicin-resistant” polymerase . promoter complexes (Fig.5) with T4 DNA as template. Yet, at a temperature significantly above the transition temperature, glycerol again does not have a significant effect on RNA synthesis in vitro. Thus 1 conclude that these compounds act in a non-specific manner by altering the conformation of the DNA template in promoter regions, in particular by allowing “opening” to occur at a lower temperature.

What is the nature of the conformational change in a promoter region? In the case of the rRNA promoter the conformational state is temperature-dependent [4]. The transition between the states is both reversible and cooperative[4]. It is probably not effected in- stantaneously, since such an instantaneous shift would be inconsistent with the results of both order-of- addition experiments (Table 2) and certain types of temperature-shift experiments [4].

To what extent are these properties consistent with some described step in the initiation of RNA synthesis at promoter sites? The sequence of molecular events necessary for the initiation of RNA synthesis by RNA polymerase holoenzyme was first formulated by Zillig and his collaborators [22] and elaborated by Bautz et al. [3] and by Hinkle and Chamberlin [21]. After unproductive random interactions with the DNA template the polymerase “recognises” a promoter site. A region of about eight base pairs at the polymerase- binding site then undergoes local strand separation [23] and the resulting polymerase . DNA complex is poised to initiate RNA synthesis very rapidly. The local melting is believed to be consequent upon recognition of a promoter nucleotide sequence by holoenzyme. The tight binding presumably provides the energy for strand separation. Thus melting will occur when the energy required for strand separation is less than the


A. Travers 441

energy made available by recognition. On this model the probability of melting and hence of initiation could be increased either by decreasing the energy required for melting or by increasing the initial affinity of the holoenzyme for the promoter site. Thus, glycerol and dimethylsulphoxide could act either by lowering the T, for melting or by altering the structure of the promoter in such a manner that its recognition parameters were changed. Both compounds are known to lower the T,,, of bulk DNA in aqueous media, although the 2 “C shift effected by 15 % glycerol [24] is substantially less than the change in transition temperature observed in transcription. Two points should be noted. First in the initiation process strand separation probably does not proceed in a completely aqueous environment but in close proximity to a protein, RNA polymerase. Secondly, since there is a requirement for strand separation at promoter sites, the melting properties of these regions might differ substantially from those of bulk DNA. Thus it appears likely that both glycerol and dimethylsulphoxide could affect transcription by lowering the melting temperature of DNA in the promoter region. Consistent with this conlusion is the observation that HI protein, which preferentially increases the T, of (G + C)-rich DNA while de- stabilising (A + T)-rich DNA (Cukier-Kahn and Geiss, personal communication) stimulates RNA synthesis from (A + T)-rich T4 promoters [25] yet increases the transition temperature between the open and closed forms of the rRNA promoters [9 ] . Nevertheless the disruption of the water envelope around the DNA, which results in the lowering of T, by glycerol and dimethylsulphoxide, could also affect the diameter of the double helix (see for example [26]). Thus I cannot exclude that these compounds may act by altering the recognition parameters of the DNA, although since their effect appears to lack specificity I consider this possibility unlikely.

In other studies on the effect of glycerol on transcription in vitro it has been shown that at concentrations up to 20% (v/v) glycerol abolishes the requirement in vitro for crp protein for transcription of the lac operon [I 11. In addition glycerol causes the h e x mutant promoter to act like a wild-type promoter, the effect of glycerol in this case being strongly temperature-dependent [ll]. If the action of glycerol in these systems is analogous to its effect on rRNA synthesis. these results imply that crp protein acts by “opening” the gal promoter and that the effect of the Asex mutation is to raise the transition temperature between the open and closed forms of the promoter. In vitro the characteristics of the control of rRNA

synthesis in vivo are paralleled most closely from the closed form of the promoter. Thus in two systems where a positive control mechanism has been invoked, i.e. catabolite repression and rRNA synthesis, the promoter must be closed for the control to be manifest.

I would like to thank Drs D. Baillie, C. Goff, B. Griffin, T. Maniatis and J. D. Smith for helpful discussions and Robin Buckland for expert technical assistance.

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A. Travers, M.R.C. Laboratory of Molecular Biology, Postgraduate Medical School, University of Cambridge, Hills Road, Cambridge, Great Britain CB2 2QH


on the nature of dna promoter conformations : the effects of glycerol and dimethylsulphoxide

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