On the action of the cyclic AMP-cyclic AMP receptor protein com - NCBI

The EMBO Journal vol.3 no. I pp.43 -50, 1984

On the action of the cyclic AMP-cyclic AMP receptor protein complex at the Escherichia coli lactose and galactose promoter regions

Annick Spassky*, Stephen Busby and Henri Buc lnstitut Pasteur, D&partement de Biologie Moleculaire, 25 rue du Docteur Roux, 75724 Paris CUdex 15, France *To whom reprint requests should be sent Communicated by H.Buc

Using DNase footprinting and transcription assays in vitro we have probed the effect of the cAMP-cAMP receptor protein complex (cAMP-CRP) on the positioning of RNA polymerase and on the location of the transcription start point at the Escherichia coli gal and lac operon regulatory regions. In both cases, RNA polymerase can form two alternative complexes which promote transcription from two different start points, SI and S2: pre-incubation of promoter DNA with cAMP-CRP results in a shift of the transcription start from S2 to S1 and in an increase in the rate of open complex formation. Moreover, the rate of formation of each heparinresistant complex parallels the establishment of the corresponding footprint, showing that the stable binding corresponds to open complex formation. We show that, in the case of gal, RNA polymerase, which is bound so as to transcribe from S2, cannot be diverted to S1 by subsequent addition of cAMP-CRP. In contrast, in the case of lac, when cAMP-CRP is added after RNA polymerase, complexes which initiate transcription at S2 are rapidly converted to complexes which initiate at S1. Finally, we present data which suggest that protein-protein interactions are essential for CRP-nduced activation at both the lac and gal promoters. Key words: cAMP receptor protein/footprinting/RNA polymerase/transcription

tion from SI is greatly reduced. However, under these conditions, most transcription starts at an alternative point, S2, 5 bp upstream of SI, a start point not found in the presence of cAMP-CRP. Hence, cAMP-CRP has a dual role, stimulating transcription from one start point whilst repressing transcription from another point (reviewed by de Crombrugghe and Pastan, 1978). Using the technique of DNase footprinting (Galas and Schmitz, 1978), we have probed the sequences in the gal promoter region which are protected by RNA polymerase either in the presence or absence of cAMP-CRP. From this analysis we conclude that the SI and S2 transcripts result from two different modes of binding of RNA polymerase. This argues for the existence of two overlapping but different promoters, P2 and P1, at which the polymerase can be engaged to transcribe from one start or the other (S2 or S1). The individuality of the two promoters is further demonstrated here by an experiment which shows that RNA polymerase bound at P2 cannot be diverted to P1 by the subsequent addition of cAMP-CRP, indicating that bound polymerase does not wobble between the SI and S2 transcription starts. Finally, measurements of the rate of formation of open complexes at P1 or P2 show that, not only does cAMP-CRP block the recognition of P2, but it also accelerates the recognition of P1. At first sight, the organisation of the lactose operon promoter region is very different from that at the galactose promoter. While the gal operon is expressed both in the presence and absence of active CRP (Perlman and Pastan, 1969; Ullmann et al., 1979), lac expression depends almost totally on the binding of cAMP-CRP 60 bp upstream from the transcription start (reviewed by Reznikoff and Abelson, 1978). This was confirmed by experiments which showed that cAMP-CRP does bind and stimulate lac transcription (Maquat and Reznikoff, 1978; Schmitz, 1981). This simple view of the lac system, which supposes that no specific interaction occurs between RNA polymerase and the lac promoter in the absence of cAMP-CRP has, however, been challenged. In particular McClure and his collaborators, after a detailed analysis of the kinetics of transcription initiation, concluded that, in the absence of cAMP-CRP, RNA polymerase could form tight-binding complexes with lac promoter DNA, which blocked the activation of the usual lac transcription start, denoted SI (Malan, 1981; McClure et al., 1982; Malan et al., 1983). Subsequently, these authors reported that, in the absence of cAMP-CRP, RNA polymerase could weakly initiate transcription at a second site, S2, 22 bp upstream from S1. Moreover, transcription from this second initiation site was found to be inhibited by cAMP-CRP. Thus, a remarkable parallel exists between the gal and lac promoters: in both cases, cAMP-CRP blocks one promoter (P2) whilst stimulating another (P1). According to this view, the major difference between the two systems is that, in the case of gal, the P2 promoter both binds polymerase and promotes transcription in vivo, whereas, in the case of lac the second pro43 -

Introduction Upon activation with the ligand, cyclic 3',5'-adenosine monophosphate (cAMP), the Escherichia coli cAMP receptor protein (CRP) can bind near the transcription start points at a number of E. coli promoters, and hence can regulate expression from these promoters (reviewed recently by Ullmann and Danchin, 1983; de Crombrugghe et al., 1983). Two of the best characterised cases are the promoters of the galactose (gal) and lactose (lac) operons. In the work reported here, using purified CRP, RNA polymerase and DNA fragments, we measured the effect of the cAMP-CRP complex on the positioning of RNA polymerase and on the rate of transcription initiation at these two promoters. The effect of cAMP-CRP at the gal regulatory region is known from the analysis of gal mRNA made both in vivo and in vitro (Musso et al., 1977; Aiba et al., 1981). When cAMPCRP binds to the gal promoter region it stimulates transcription from a specific start point, S1. Structural and genetic studies have identified the CRP binding site at 35 bp upstream from SI (Taniguchi et al., 1979; Busby et al., 1982) and stoichiometry measurements have shown that one CRP dimer binds at this locus (Shanblatt and Revzin, 1983; Kolb et al., 1983). In the absence of the cAMP-CRP complex, initia-

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Transcription in vitro from the lactose and galactose operon promoter regions. The left-hand panel of the figure shows an autoradiogram of a gel on have analysed transcripts from an 890-bp Pstl-HindIII fragment containing the gal promoter region. In lanes a e the fragment was incubated with

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nucleoside triphosphates and heparin. In lanes f -h, CRP (g) or 20 min (h) before the initiation of transcription. As the gal promoters are located near the Hindlll end of the fragment the run-off gives discrete transcripts 47 -52 bases long; their origins are indicated on the schematic representation of the gal promoter region drawn after Taniguchi et al. (1979). The bar, representing the DNA, is numbered with respect to the CRP-dependent start (SI). The locations of the corresponding CRP site and Pribnow box are shown by the hatched bars. Above we show the location of the CRP-independent transcription start (S2) and, the corresponding Pribnow box (Pb) and '- 35 region'. The right-hand panel of the figure shows an autoradiogram of a gel on which we have analysed transcription from a 203-bp EcoRI-EcoRI fragment containing the lac promoter region. In lanes n r CRP was first added: the fragment was then incubated with RNA polymerase for 30 s (n), 2 min (0), 8 min (p), 20 min (q) or 60 min (r) prior to the addition of nucleoside triphosphates and heparin. In lanes i m, no CRP was used and the fragment was incubated with polymerase for 30 s (i), 2 min (), 8 min (10, 20 min () or 60 min (in) before the initiation of transcription. The run-off transcription give bands 65 -87 bases long: the origins are indicated oni the schematic representation of the lac promoter region, drawn after Malan (1981). RNA

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moter binds polymerase but can only weakly promote transcription. To confirm the existence of a second promoter in the lac system, in parallel with the studies with gal DNA, we have investigated the effect of cAMP-CRP on the positioning of RNA polymerase at the lac promoter region. Additionally, again in parallel with the gal system, we have analysed the transcripts made both in the presence and absence of cAMPCRP. We conclude that, indeed, like the gal system, the lac system contains a promoter which operates in the absence of cAMP-CRP and which gives rise to a transcript longer than the one found in the presence of cAMP-CRP. Kinetic studies using transcription and footprinting tests, show that in the lac system, unlike in the gal system, cAMP-CRP can easily displace RNA polymerase from P2 to P1.

44

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Results

Run-off transcription at the lac and gal promoters To study the effect of cAMP-CRP on in vitro transcription from the lactose and galactose promoters, we have used the 'run-off' method (Materials and methods). Figure 1 shows gels on which transcripts were analysed after gal or lac promoter fragments had been incubated with RNA polymerase either in the presence or absence of cAMP-CRP. In the case of galactose, we confirmed that the transcript seen in the absence of CRP (lanes a - e) is longer than that in its presence (lanes f-h). Calibration of the gels showed that the two transcripts differed in length by five nucleotides, as expected from the work of Musso et al. (1977) who had mapped the position and direction of the two transcripts on the

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Fig. 2. Footprint analysis of the lac and gal promoters. The figure shows a series of autoradiograms of gels in which the pattern of digestion of various endlabelled fragments in the presence of various combinations of CRP and polymerase was analysed. The combinations of CRP (100 nM) and polymerase (150 nM) are indicated by the '-' or '+' symbols above each lane. When CRP and polymerase were both present the CRP was added before the polymerase. Lanes a d show analysis of the lower strand at the lac promoter whilst lanes e h show analysis of the upper strand. Lanes i I show analysis of the lower strand at the gal promoter whilst lanes m p show analysis of the upper strand. The calibrations shown in the figures were deduced by running sequence reactions on the same gels: the calibration is with respect to the start point for the CRP-dependent transcription start. -

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gal sequence (shown schematically in the centre of Figure 1). A similar experiment with a fragment containing the wildtype lac promoter showed, in the presence of CRP (lanes n r), a cluster of bands which, from the calibration, must originate from a group of start points - 65 bp from the end of the fragment, a zone which contains the + 1 position, as defined by Reznikoff and Abelson (1978). In the absence of CRP (lanes i m) although less transcription initiates in the zone, a new transcript is observed, which had been almost totally absent in the presence of CRP. This transcript (marked S2 in the Figure) which starts 85 -87 bp from the end of the fragment can be identified with the second lac transcript found previously (Maquat, 1978; Malan, 1981; McClure et al., 1982). The positions of the two lac transcription start points, shown schematically in Figure 1, were further confirmed by experiments in which transcripts were marked with different -y-labelled nucleoside triphosphates allowing the identification of the 5' nucleotide. In the presence of cAMPCRP the transcripts initiated at positions from + 1 to -6 whilst in its absence, initiation was at -21 or -22 (A.Spassky, unpublished data). In such transcription experiments, apart from bands cor-

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responding to the S2 and SI starts, we also observed high mol. wt. bands, which, from the calibration, corresponded to transcripts longer than the DNA fragment used. Kinetic experiments (see below, Figure 3) show that their appearance parallels that of the S2 and SI bands. We conclude therefore that they are most likely due to S2 or SI transcripts which are either aggregated or elongated (see Zubay, 1980): their presence does not affect the conclusions we reach in the Discussion.

Footprint analysis of RNA polymerase binding in the absence of cAMP-CRP Figure 2 a-h shows the pattern of protection on the upper and lower strand at the lac promoter. RNA polymerase induces a weak protection of bands from 46 to around + 1 (lanes b and f). In contrast, and in agreement with Schmitz (1981), we find that cAMP-CRP alone protects the neighbouring zone from -48 to -74 (lanes d and h). However protection is not total and a number of bands in this zone are enhanced (- 57 and -66 on the lower strand and 58 and -67 on the upper strand). Finally, in the presence of both cAMP-CRP and polymerase, protection stretches from 74 45

presence or

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to + 16 (lanes c and g). Although this zone includes the regions covered by both CRP and polymerase alone, the protection is stronger and more extensive. However, digestion at several positions in this zone is enhanced, including, notably, those which were enhanced by the binding of cAMP-CRP

alone. Similarly, Figure 2 i - p shows the pattern of protection with the same proteins at the gal promoter. In agreement with Taniguchi et al. (1979, 1983), with cAMP-CRP alone we observe weak protection between - 30 and - 52 as well as the enhancement of a number of bands in this zone (lanes 1 and p). In contrast, with RNA polymerase alone, digestion is blocked from + 10 to - 20 whilst some bands are protected as far as -41 (lanes j and r). Finally with both cAMP-CRP and polymerase, as noted by Shanblatt and Revzin (1983) and Taniguchi et al. (1983), the protection is more extensive, stretching from - 67 to + 19 (lanes k and o). As with lac, the protected zone contains a small number of positions at which digestion is enhanced: again these include the positions at which cAMP-CRP alone increased digestion (at -35 and - 47 on the lower strand and at - 45 on the upper strand). The rate offormation of transcriptionally active complexes The results demonstrate that at the gal and lac promoters the transcription start point varied according to the presence or absence of cAMP-CRP, as does the zone protected by RNA polymerase. We have measured the rate of initiation of transcription from P2 and P1, using the same assay as described in Figure 1. At various times after the addition of polymerase to promoter DNA, heparin and radioactively labelled nucleoside triphosphates were added and the transcripts made were analysed on a gel. After quantification of the two transcripts, we could measure the rate of formation of complexes capable of promoting P2 or P1 transcription at either promoter (Figure 3A and B). At the gal promoter in the absence of cAMP-CRP, the complex responsible for transcription from S2 takes several minutes to form (Figure 3A, squares). Note that a small amount of SI transcript is seen without cAMP-CRP and that the complex responsible for this also forms slowly (Figure 3A, triangles). In contrast, in the presence of cAMP-CRP, transcription from S2 is inhibited but transcription from SI occurs too rapidly to measure by this method (Figure 3A, circles). Figure 3B shows similar experiments at the lac promoter in the presence of cAMP-CRP (circles). Complexes which can transcribe from P1 form with a half-time of -2 min whereas, in the absence of cAMP-CRP, the half-time for the formation of complexes from P2 is >20 min (squares). We conclude that the rate of formation of different open complexes differs according to the presence or absence of cAMP-CRP. In a parallel series of experiments, using the same concentrations of reagents, we have measured the time for the establishment of the footprints of RNA polymerase at P2 and P1 for both gal and lac. To do this, samples were exposed to DNase I digestion, as in Figure 2, at different times after mixing with RNA polymerase. From the intensity of the footprint, the kinetics of establishment of each complex was deduced. The results (Figure 3C and D) show that the establishment of the footprint parallels, within experimental error, the kinetics of formation of complexes capable of transcription from either P2 or PI at both gal and lac. We infer that the footprint patterns shown in Figure 2 are true reflections of the complexes which are formed on transcrip46

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Fig. 3. Panels A and B show the kinetics of formation of complexes which can transcribe from P1 or P2 at the gal or lac promoter regions. Transcriptions were carried out as shown in Figure 1. After autoradiography, films were scanned and the relative intensity of the transcripts from PI or P2 was determined after densitometry. The intensity of each band is plotted as a function of the time of pre-incubation of DNA with RNA polymerase. Panel A shows an experiment with gal DNA: we show the formation of complexes which can transcribe from P2 (O) or P1 (A) in the absence of CRP and the formation of complexes which initiate transcription from P1 in the presence of CRP (0). Panel B shows an experiment with lac DNA showing the transcription from P2 (O) or P1 (A) in the absence of CRP and transcription from P1 in the presence of CRP (O). Panels C and D show the time course of formation of polymerase-DNA complexes at the gal and lac promoters as judged by the establishment of the footprint in protection experiments (see text and Materials and methods). Panel C shows the time course of the changes at the lac promoters in the absence (El) or presence of CRP (0). In the absence of CRP we show the change in the band at -47 normalised against that at + 19, which is unchanged. We obtained similar results following the bands at -20, -10 or +9. In the presence of CRP, the data show the changes at -47: identical results were obtained with the band at + 13. Panel D shows the time course of the changes at the lac promoter in the absence of (E) or presence of CRP (0). In the absence of CRP we plot the data for the band at - 18 (the bands at + 17, - 10 and -48 give identical results), and in the presence of CRP, we plot data for the band at - 10 (the band at -25 gives a similar result). In both cases the intensities were normalised on the band at +28 which does not change.

tion initiation. The interconversion of RNA polymerase from P2 to P1 To measure the interconversion from P2 to P1, DNA was pre-incubated with RNA polymerase before addition of cAMP-CRP. Figure 4A shows a gel analysis of such an experiment in which we analysed transcripts from the lac promoter region. Lane a shows the transcripts made after preincubation of promoter DNA with RNA polymerase. The transcripts made after different times of incubation with cAMP-CRP are seen in lanes b - f. Even after the shortest time (lane b, 30 s) transcription from S2 is almost totally blocked whilst the stimulation of S1 takes place more slowly (lanes b - f). For comparison we performed a parallel experi-

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Fig. 4. The effect of the order of addition of CRP and polymerase on the transcription and footprint patterns at the lac and gal promoter regions. The figure shows autoradiograms of gels on which transcripts from the lac and gal promoter regions (panels A and C) have been analysed or on which the footprint pattern of combinations of CRP and polymerase have been determined (panels B and D). Panel A: lac promoter DNA was incubated with RNA polymerase (lane a) followed by CRP for different times (b f). Transcription was then started with nucleoside triphosphates and the transcripts were analysed. Lanes g k show a similar experiment in which the DNA was pre-incubated with CRP prior to incubation with polymerase for different times. The positions of transcripts starting at SI and S2 are marked. Panel B: footprint analysis of lac DNA incubated with different combinations of CRP and RNA polymerase. The various combinations are indicated above each lane: '-' indicates no addition, '1' indicates the first product added and '2' shows the second addition. The concentration and conditions used were exactly as for the transcription experiment: each component was incubated for 20 min before the DNase attack. The calibrations shown were deduced by comparison with sequencing gels. Panel C: gal promoter DNA was pre-incubated with RNA polymerase (lane a), CRP and then RNA polymerase (lanes b- d) or RNA polymerase then CRP (lanes e- h) before initiation of transcription by adding nucleoside triphosphates and heparin. Whilst the first protein was incubated for 20 min, the second addition was incubated for different times, which are shown above each lane. The arrows show the position of transcripts originating at the S2 or SI transcription start points. Panel D: footprint analysis of gal DNA incubated with different combinations of CRP and RNA polymerase. -

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ment in which cAMP-CRP was added before the addition of polymerase (lanes g k); the rate of formation of the open complex at P1 is unaffected by whether or not P2 was previously occupied. We performed similar experiments with gal DNA. Figure 4C, lane a, shows the transcription from the S2 start after pre-incubation with RNA polymerase. However, when cAMP-CRP is subsequently added (lanes e-h) RNA polymerase is hardly displaced from the P2 open complex and a trace of transcription from the SI start appears only after 1 h (lane h). We conclude that whilst cAMP-CRP can easily displace polymerase from lac P2, at gal P2 open complexes are more resistant. We confirmed this by footprint experiments where we examined the protection patterns of samples from the transcription experiments. At lac the footprint observed when cAMP-CRP is added after polymerase (Figure -

4B, lane c) resembles that seen when open complexes at P1 formed by adding cAMP-CRP and then polymerase (lane d) rather than that seen in the presence of polymerase alone (lane b). Similar experiments were done with the gal promoter region (Figure 4D). Lanes b and d respectively show the protection pattern when RNA polymerase or CRP and polymerase are installed at P2 or P1. Lane c shows the protection pattern when polymerase at P2 is challenged for 20 min with cAMP-CRP. The extensive protection associated with P1 open complexes does not arise, confirming the result of the transcription experiment. However some distinct changes occurred at the same positions as those affected by cAMP-CRP alone (for example the enhancements at -47 and 35 on the lower strand). From this we conclude that, when added after RNA polymerase, cAMP-CRP can bind to are

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Fig. 5. The effect of promoter mutations on footprint and transcription patterns at the gal and lac promoter regions. Panel A shows autoradiograms of gels on which we have analysed the pattern of digestion of fragments containing mutant gal promoters in the presence of various combinations of CRP and polymerase. Lanes a - e show an experiment performed with DNA containing the p37 mutation, lanes g -k show DNA with the R503 deletion and lanes I - p show the wild-type promoter as a control. The various combinations of polymerase and CRP used are shown above each lane. 'I' indicates the first product added and '2' indicates the second. The DNA was incubated with each component for 20 min before addition of DNase 1. The calibration shown in the figure was deduced from sequencing reactions (lanes f and q). Panel B shows autoradiograms of gels on which we have analysed the transcripts made after various combinations of RNA polymerase and CRP were incubated with DNA containing mutant gal promoters. The incubation conditions prior to the addition of nucleotide triphosphates were exactly as in the footprinting experiments: above each lane the order of addition of CRP and polymerase is indicated. In lanes b - d the DNA contained the p37 mutation, in lanes e -g it carried the R503 deletion whereas in lanes h -j the wild-type promoter was used as a control. Lane a shows a sequencing reaction which was used to calibrate the gel and deduce the position of the S2 and Sl starts.

its site but cannot displace RNA polymerase from the open complex at P2.

Interactions of RNA polymerase and CRP at mutant galpromoters We have previously isolated a series of gal promoter mutants

which specifically block the activation of P1 because they stop the binding of cAMP-CRP (Busby et al., 1982, 1983; Kolb et al., 1983). We have examined the interactions of polymerase with gal promoter DNA containing two such mutations: the p37 mutation, a GC to AT transition at - 37 and the R503 deletion, which removes the gal sequence between -45 and -92. Figure 5A shows a footprint analysis whilst Figure SB shows the run-off transcripts made in vitro: in both cases we have compared the behaviour of the p37 mutation and R503 deletion with that of the wild-type gal promoter. The footprint analysis shows that, in the absence of cAMP-CRP, RNA polymerase forms apparently identical 48

complexes at the P2 promoter with DNA containing either the wild-type or mutant DNA (Figure 5A, lanes b, h and m); strong protection is seen between + 10 and -20 and weaker protection is seen up to - 40. In contrast, differences are seen in the binding of cAMP-CRP, which by itself changes the footprint pattern at the wild-type promoter (Figure 5A, lane p) causing, for example, enhancements at - 35 and - 47, but has no influence together with either of the two mutants (Figure SA, lanes e and k). Surprisingly, in the presence of RNA polymerase, cAMP-CRP-induced changes are seen at the mutant promoters (lanes c and d, i and j). To explain this we suggest that while the mutations weaken CRP binding in the presence of polymerase, the weak interaction between CRP and its site must be stabilised. Figure 5A shows that if cAMP-CRP is added after polymerase is first installed at P2, changes occur at the mutant promoters similar to those seen with the wild-type promoter: for example, small enhancements of the bands at -35 and ..,47ere seen, signalling the binding of CRP. CRP appears to

Interactions of RNA polymerase at lac and gal promoters

bind but does not stop transcription from P2 (Figure 5A, lanes c, i and n, and SB, lanes c, f and i). In contrast, if CRP is added before polymerase, more radical changes are observed: some protection is seen in the zone upstream of - 48 and between - 20 and - 34 (Figure 5A, lanes d and j). This suggests that, while most polymerase remains at P2, a small number of polymerase molecules are guided to P1. This is confirmed by transcription experiments which show that when cAMP-CRP is added before RNA polymerase (Figure 5B, lanes d and g), a small switch from P2 to P1 transcription occurs at both mutant promoters. To expain this we suggest that, for promoters carrying the p37 mutation or R503 deletion, the small number of cAMP-CRP molecules which bind before polymerase addition can be stabilised by the subsequent binding of polymerase at P1. Hence the presence of polymerase allows binding of cAMP-CRP in cases where it would otherwise not stably bind. Discussion Our results clearly confirm the existence, at both the gal and lac regulatory regions, of two overlapping promoters, P1 and P2, modulated by cAMP-CRP. In both cases cAMP-CRP stimulates transcription from a start point (S1) downstream from the start point in its absence (S2). Changes in the footprint due to RNA polymerase are closely correlated with changes in the transcription start point: discrete footprints are observed when polymerase transcribes from one or the other starts. Furthermore, the rate of establishment of different footprints agrees well with the formation of different complexes capable of transcription from one promoter or another. From these observations, we infer that, in each case, the stable positioning of polymerase seen by the footprint reflects the formation of the heparin-resistant open complex. The overall picture at the gal and lac promoters thus appears similar. However, our study reveals some important differences: notably, open complexes formed in the absence of cAMP-CRP are less stable in the case of lac than in the case of gal. They give a much weaker footprint and are slower to form. Furthermore, RNA polymerase is rapidly displaced from these complexes if cAMP-CRP is subsequently added. In contrast with gal, the corresponding complexes are stable. The instability of polymerase at the lac P2 promoter could be due either to dissociation of the polymerase from the DNA or to displacement on the DNA. We think the latter mechanism is more likely since Malan (1981) has shown that polymerase has a long residence time at the lac promoter region even in the absence of cAMP-CRP. The effect of cAMP-CRP at both regulatory regions is to displace the transcription start point downstream 20 bp in the case of lac and 5 bp in the case of gal, and to sharply accelerate the formation of open complexes. The footprint analysis shows that CRP and RNA polymerase form ternary complexes at both gal and lac. Moreover, in these complexes, CRP appears to bind at the same position as in the absence of polymerase. The problem is to understand how CRP directs polymerase to the P1 complex, and why, only in the case of gal, when CRP is added after polymerase, it binds but fails to activate P1. We suggest that this difference results from a conjunction of two elements: the weakness of lac P2 and the different relative disposition of the CRP and polymerase binding sites at lac and gal. In the case of lac, at P1, the CRP and polymerase binding sites are adjacent whereas, at P2, the polymerase ' - 35 binding region' overlaps the CRP si A -

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noted above, polymerase is not tightly anchored at P2. We suggest that this promoter is, in a sense, 'imperfect': it does not allow a unique flawless fit with the polymerase, which is continually shuffled between a series of complexes in equilibrium with that responsible for the S2 start. Hence transient dissociation from the '- 35 region' may allow CRP to recognise its site. Thus it could subsequently provide the protein-protein contacts necessary to slide the polymerase by 20 bp, two turns of the helix, to the P1 complex. In contrast, in the case of gal, the binding of polymerase at P2 is strong and irreversibly commits the polymerase to transcribe from S2. Surprisingly in this case, CRP can still bind at its site without hindrance from the polymerase. However, we speculate that the potential protein-protein contacts between polymerase and CRP are out of phase (by 5 bp or 180°) and consequently no switching of the polymerase to P1 is possible. On the other hand, if CRP is added first, it directs polymerase to an alternative position, the P1 complex, because the protein-protein contacts provide more stabilisation than that due to independent binding of polymerase at P2 and CRP at its site. Though unproven, the view that CRP directs the binding of RNA polymerase by protein-protein interactions is supported by two lines of evidence. Firstly, in the presence of RNA polymerase, some stable CRP binding can be detected at mutant gal promoters which cannot stably bind cAMPCRP alone. In these cases some P1 transcription is observed in vitro. Hence polymerase and CRP mutually stabilise each other in P1 open complexes. Secondly, Shanblatt and Revzin (1983) have suggested that the presence of polymerase at P1 induces the binding of a second CRP molecule between - 50 and - 70 upstream of the first. As the precise sequence of this zone is not important for this supplementary binding, it appears to be dependent on protein-protein contacts. The effect of CRP on the rate of formation of P1 open complexes further underlines the active role of CRP at P1. The clear acceleration in the rate shows that CRP acts positively on the kinetics of P1 complex formation, rather than simply blocking access to P2, a competing site. However, CRP directs polymerase to a tight discrete complex at both gal and lac, but, in the case of lac P1, there is considerable ambiguity in the exact transcription start point. Under our conditions, the transcript can start at any of six bases around the position labelled '+ 1' by Reznikoff and Abelson (1978). This type of ambiguity, which has been previously observed by Gralla and his colleagues at mutant lac promoters, is best explained by supposing that the singlestranded template at the polymerase active site may diffuse to a number of alternative start bases in the active site (Carpousis et al., 1982). It is interesting that this ambiguity appears to be independent of the strength of binding of polymerase to the promoter: thus, we observe this effect at the tight lac P1 complexes, but the initiation point seen with the weaker lac P2 complexes is more precise. Furthermore, it is important to note that the alternance between gal S1 and S2 cannot be due to such a mechanism in which CRP would bias the position of the template strand at the polymerase active site. The data clearly show that, for gal, CRP cannot push the start point from S2 to S1 and, in any case, polymerase is bound differently in complexes which transcribe from one start or another. A number of important differences between the gal and lac systems have emerged from these in vitro studies. As noted in

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the Introduction, a further major difference is the role of the second promoter in vivo. In the case of gal, P2 has an important activity in gal expression in vivo whereas, in the case of lac, the second promoter appears to be used as a polymerase binding site to block the expression of the first promoter. In the case of gal, the system has evolved so as to assure the expression of gal enzymes when the level of cAMP-CRP is reduced, whereas in the case of lac the system has been adopted to minimise expression in this condition. Clearly, the operation of overlapping and mutually exclusive promoters can generate a considerable number of different regulatory

patterns. Materials and methods DNA fragments

By convention, lac and gal sequences are numbered from the transcription point of the cAMP-CRP dependent promoter. Sequences upstream of this point are labelled with a prefix and sequences downstream are labelled with a '+' prefix. Plasmid DNA was isolated from host bacteria using the SDS method described by Maniatis et al. (1982). After restriction the appropriate fragments were isolated using the electroelution method described by these authors or the start

analysis on 10% sequencing gels, run as above and developed by autoradiography. The gels were calibrated with sequence reactions performed on the same labelled fragments. From the calibration we deduced the positions of protected or enhanced bands for each set of conditions. By convention, throughout the text, we refer to a change at any particular base as meaning an alteration in the cleavage of the phosphodiester bond on the 3' side of the base. To quantitate the data from footprints, autoradiograms were scanned on a Vernon densitometer and the intensity of individual bands was calculated by integration. The extent of protection or enhancement at any band was calculated after normalising the intensity against that of bands which did not change. These variations were then expressed on a 0- 100%o scale defined by the intensity of the band before and after the studied change. Isotopes, enzymes and reagents ['y-32P]ATP, [la-32P]dATP and [a-32P]UTP were obtained from Amersham. Restriction enzymes, Klenow fragment and polynucleotide kinase were purchased from Boehringer or BRL Ltd and used under the conditions specified by the supplier. DNase I was obtained from Worthington. Other reagents were of the highest quality available.

Acknowledgements We are grateful to Denise Kotlarz for help in the early stages of this work and to Odile Delpech for the artwork. This work was supported by grants no. 955112 and 955171 from the Centre National de la Recherche Scientifique.

'crush and soak' method of Maxam and Gilbert (1980). DNA containing the lactose operon promoter region was isolated on an EcoRI-EcoRI 203-bp fragment cloned in a pBR322 derivative (Schaeffer et al., 1982). This fragment covered the lac sequence from - 140 to + 63. For the footprint experiments this fragment was labelled with ['y-32P]ATP by polynucleotide kinase or with [cr-32P]dATP by Klenow fragment DNA polymerase. Labelled fragments were subsequently cleaved with PvuII which cuts the fragment at - 123 and generates promoter fragments uniquely labelled either on the upper or lower strand. DNA containing the galactose operon promoter region was isolated on an 890-bp Pst-HindlII fragment isolated from pAA187 (Busby and Dreyfus, 1983). This fragment contained the 750-bp Pst-EcoRI fragment from pBR322 joined to a 144-bp EcoRI-HindIII gal promoter fragment containing the gal sequence from - 92 to + 45. The upper or lower strands were labelled by incorporating 32p label at either the EcoRI or HindIII sites with polynucleotide kinase. Parallel preparations were performed using DNA containing the p37 point mutation at - 37 (Busby and Dreyfus, 1983) or the R503 deletion which removes the gal promoter sequence from -46 to -92 (Busby et al., 1983).

Galas,D. and Schmitz,A. (1978) Nucleic Acids Res., 9, 3157-3170. Kolb,A., Busby,S., Herbert,M., Kotlarz,D. and Buc,H. (1983) EMBO J., 2,

Proteins

Lowe,P.A., Hager,D.A. and Burgess,R.R. (1979) Biochemistry (Wash.), 18,

E. coli RNA polymerase was prepared according to the modified procedure of Burgess and Jendrisak (Lowe et al., 1979). The concentration of enzyme was

calculated according to these authors after absorption measurements. Purified CRP was kindly given by Bernadette Blazy. It was > 98% pure as judged by SDS-polyacrylamide gel electrophoresis. Its concentration was determined spectrophotometrically using A278 = 4.1 x 104/M/cm per CRP dimer (Takahashi et al., 1982).

Transcription experiments 1-2 nM DNA fragments were incubated at 37°C in the presence or absence

of 100 nM CRP and 200 uM cAMP in standard buffer (final concentration 40 mM Tris pH 8, 10 mM MgCl2, 100 mM KCI, 0.1 mM dithiothreitol, 5% glycerol). After 20 min, 150 nM RNA polymerase was added: at different times 90 Al aliquots were taken and added to 10 tl of a cocktail containing 3 mM ATP, GTP and CTP, 1 mM UTP, 3 1Ci [32P]UTP and 1 mg/ml heparin. After a 15 min incubation at 37°C the elongation reactions were stopped by addition of 0.3 M sodium acetate, 30 isg/ml tRNA and 1 mM EDTA and the samples were extracted with phenol, precipitated with alcohol, rinsed, and dried prior to loading on a 10%o sequencing gel, made and run according to Maxam and Gilbert (1980), and calibrated with sequence reactions as described by Aiba et al. (1981). In some cases RNA polymerase was added before cAMP-CRP. In experiments where no time course was determined, polymerase and/or CRP were each pre-incubated with DNA for 20 min before transcription was started by adding the cocktail of triphosphates and heparin.

Footprint experiments End-labelled DNA was incubated with the same concentration of polymerase and cAMP-CRP in the same buffer and for the same time as the transcription experiments. The order of addition of CRP and polymerase was permuted as above. The footprint was determined by adding 75 ng/ml DNase I to 100 Al aliquots from these mixtures and by incubating (at 37°C) for 18 s. Digestion was stopped by the addition of 0.3 M sodium acetate, 30 ig/ml tRNA and 0.1 mM EDTA followed, by phenol extraction, ethanol precipitation and

50

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Ullmann,A., Joseph,E. and Danchin,A. (1979) Proc. Natl. Acad. Sci. USA, 76, 3194-3197. Ullmann,A. and Danchin,A. (1983) Adv. Cyclic Nucleotide Res., 15, 1-53. Zubay,G. (1980) Methods Enzymol., 65, 856-877. Received on 22 August 1983