molecular interplay between rna polymerase and two transcriptional regulators in promoter switch

Molecular Interplay Between RNA Polymerase andTwo Transcriptional Regulators in Promoter Switch

Ana Camacho and Margarita Salas*

Instituto de Biologıa Molecular“Eladio Vinuela” (CSIC)Centro de Biologıa Molecular“Severo Ochoa” (CSIC-UAM)Universidad Autonoma, CantoBlanco, 28049 Madrid, Spain

Transcription regulation relies in the molecular interplay between theRNA polymerase (RNAP) and regulatory factors. Phage f29 promotersA2c, A2b and A3 are coordinately regulated by the transcriptional regula-tor protein p4 and the histone-like protein p6. This study shows that pro-tein p4 binds simultaneously to four sites: sites 1 and 2 located betweenpromoters A2c and A2b and sites 3 and 4 between promoters A2b andA3, placed in such a way that bound p4 is equidistant from promotersA2c and A2b and one helix turn further upstream from promoter A3.The p4 molecules bound to sites 1 and 3 reorganise the binding of proteinp6, giving rise to the nucleoprotein complex responsible for the switchfrom early to late transcription. We identify the positioning of the aCTD-RNAP domain at these promoters, and demonstrate that the domains arecrucial for promoter A2b recognition and required for full activity of pro-moter A2c. Since binding of RNAP overlaps with p4 and p6 binding,repression of the early transcription relies on the synergy of the regulatorsable to antagonize the stable binding of the RNAP through competitionfor the same target, while activation of late transcription is carried outthrough the stabilization of the RNAP by the p4/p6 nucleoprotein com-plex. The control of promoters A2c and A2b by feed-back regulation isdiscussed.

q 2003 Elsevier Ltd. All rights reserved.

Keywords: DNA-footprinting; feed-back regulation; promoter-regulation;RNA-polymerase; transcriptional-regulators*Corresponding author

Introduction

Regulation of transcription initiation is a key stepin the control of gene expression. In eukaryotes,transcription regulation often involves co-operativebinding of multiple transcriptional regulatoryproteins. In bacterial RNA polymerase-dependentpromoters, few mechanisms of co-dependence ontwo or more factors functioning synergistically havebeen identified to date, most prokaryote transcrip-tion regulation systems described relying on a singleregulator. A fundamental question of gene regu-lation in both eukaryotes and prokaryotes wheretwo or more factors are involved is how the inter-action between the proteins regulates differentialgene expression.

The prokaryotic mechanisms by which promoterregulation can be set by multiple regulators have

been subjected to intense scrutiny, and both antag-onism and synergism between transcriptional reg-ulators have been described. In Escherichia coli, theantagonism and co-operativity between FIS andCRP for transcription regulation has been docu-mented at several promoters, among them the crp,fis, bgl and proP promoters. FIS and CRP modulatepromoter expression by altering the compositionof functional nucleoprotein complexes formed inconjunction with RNAP at the promoter regulatoryregion.1 – 4 Examples of synergy between factors canbe found in the activation of the malE promoter orin the repression of the cytR regulon. Activation ofmalE relies on the formation of a higher-orderstructure involving co-operative binding of MalTto promoter distal sites, as well as CRP binding inbetween the bound MalT. MalT is the primary acti-vator and one function of CRP is to facilitatebinding of MalT to its cognate sites by bendingthe intervening DNA.5 The expression of promo-ters of the cytR regulon is regulated after theformation of a ternary complex composed of CRP-CytR-CRP.6 – 8 An additional example of synergism

0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

E-mail address of the corresponding author:[email protected]

Abbreviations used: RNAP, RNA polymerase; aCTD,RNAP a-subunit C-terminal domain.

doi:10.1016/j.jmb.2003.12.039 J. Mol. Biol. (2004) 336, 357–368

between two transcription regulators is the switchfrom early to late transcription of Bacillus subtilisphage f29.

Phage f29 gene expression takes place from fivemajor promoters, A1, A2b, A2c, A3 and C2. Threeof these promoters, early promoters A2c and A2band late promoter A3, are clustered within 219 bp(Figure 1A) and are regulated coordinately by amultimeric complex of the early viral proteins p4and p6 that elicits the switch from early to latetranscription, repressing promoters A2c and A2band activating promoter A3.9 – 11 However, the pre-cise binding positions of proteins p4 and p6, andhow the interplay between these proteins andRNAP at the promoter sequences yields the differ-ential regulation needed for the switch from earlyto late transcription are unknown. Our goal is tounderstand how the functional p4/p6 complex isformed and how this complex differentiallycontrols promoter transcription. In this work, wedescribe the arrangement of proteins p4 and p6 onthe DNA sequence encompassing promoters A2c,A2b and A3 and we study the positioning ofRNAP when bound at these promoters. The resultssuggest that the viral switch between early and latetranscription results from the interplay betweenRNAP and the phage-encoded regulatory proteinsp4 and p6 through competition for overlappingbinding sites driving to the alteration in the com-

position of the nucleoprotein complex bound topromoters A2c, A2b and A3.

Results

Positioning of proteins p4 and p6 to form atranscriptional regulatory complex

To study the interaction of proteins p4 and p6with DNA, we have used hydroxyl radical foot-printing, since it allows determination of thepositions of the DNA backbone contacted bythe protein with a high degree of resolution. TheDNA sequence under study comprises 364 bpfrom position þ77 (coordinate 4895) of promoterA2c to position þ68 (coordinate 5257) of promoterA3 (Figure 1A). In several cases, due to technicalrequirements, the DNA was labelled near a par-ticular point of the sequence; however, all exper-iments where the p4/p6 nucleoprotein complexwas analysed included the entire protein p4 bind-ing region from position þ42 (coordinate 4930) ofpromoter A2c to position 225 (coordinate 5165) ofpromoter A3. Thus, fragments containing promo-ters A2c, A2b and A3, labelled in the upper orlower strand, were incubated with protein p4, pro-tein p6 or both and subjected to hydroxyl radicalfootprinting. The results are shown in Figure 1

Figure 1. Analysis by hydroxyl radical footprinting of binding of proteins p4 and p6 to the sequence including pro-moters A2c, A2b and A3. A, Scheme showing the location of early promoters A2c and A2b and late promoter A3. The210 and 235 elements for B. subtilis sA-RNAP at each promoter and previously described p4 binding regions are indi-cated. Several nucleotide positions mentioned here are shown. B–E, Hydroxyl-radical footprinting of the binding ofproteins p4 or/and p6 to DNA sequences from positions 4930p to 5218 (B), from 5165p to 4895 (C), from 5218p to 4930(D) and from 4895p to 5165 (E) where the asterisk represents the location of the label. P4 and p6 were present at300 nM and 12.5 mM, respectively. When present at several concentrations, p4 was at 35, 75, 150 and 300 nM and p6at 2.5, 6, 12.5 and 24 mM. Protein p4 contact positions 10 –120 are indicated at the left of each panel. Promoter startsites indicated by arrows and some positions relative to the promoters start site are depicted to the right. Several pro-tected nucleotide positions are denoted by vertical bars.

358 Transcriptional Regulators and Promoters Switch

and a summary of the patterns of protection onboth DNA strands is shown in Figure 2.

Protein p4 binding results in the protection of sixsets of bands separated by about 10 bp (protectionsites 10 –60; Figure 1B and C, lanes b) between pro-moters A2c and A2b, and of another six sets ofbands equally separated between promoters A2band A3 (protection sites 70 –120; Figure 1D, lane band E, lanes b–d). The only exception to this regu-lar pattern is the 13 nucleotide spacer between pos-itions 40 and 50 in the upper strand (Figure 1C,lanes b). Not all p4 contacts seem to have equalbinding affinity; when increasing concentrationsof p4 were added (Figure 1E, lanes b–d) protec-tions 70 –90 were detected first while detection ofprotections 100 and 120 required greater amountsof p4. Protections 10–60 were also detected at alower concentration of p4 than those at positions100 –120 (not shown). An additional protection, atabout the middle point between the two sets of sixbands and centred at position 29 of the promoterA2b start site (Figure 1C, lanes b) should be noted.As shown in Figure 2A, protein p4 contacts withthe same periodicity both DNA strands but dis-placed 1–3 bp in one strand with respect to theother, indicating binding of the protein to one faceof the DNA helix. Protections 10 –30 are located atthe p4 binding site 1 (Figure 2A) and protections40 –60 overlap with p4 binding site 2, described asthe site occupied by p4 in the presence of RNAP;12

however, our data indicate that p4 binds to site 2also in the absence of RNAP (Figure 1C, lanes b).Protections 70 –90 are located at p4 binding site 3(Figure 1D, lane b),13 which was described as aninverted repeat with a 15 bp spacer (50-CTTTTT-15bp-AAAATG -30). Hereby, we define protein p4binding site 4 encompassing protections 100–120

that includes the imperfect inverted repeat50GTTTTT-15bp-CAAATC 30 (Figure 2A).

The hydroxyl radical footprinting of p6 showssets of protections of three to five nucleotidesspaced regularly every 11–13 nt expanding theentire promoters-containing fragment (Figure 1Band D, lanes f, and C, lanes c) with two exceptions;one around position þ20 of promoter A2b (Figure1C, lanes c) where the spacer of the p6 protectionsis only of 9 bp in both strands (see also Figure 1D,lanes f), and the other between positions 224 and247 of promoter A2b, where two contiguousspacers in the lower strand (Figure 1D, lane f) areof 7 nt and 15 nt, while the 12 nt spacer is main-tained on the upper strand (see also Figure 2B).Independent of the length of the fragment ana-lysed, p6 contacts identical nucleotide positions,indicating binding specificity in the absence of aconsensus sequence (Figure 2B).

Analysis of the binding locations of proteins p4and p6 within the multimeric nucleoprotein com-plex was carried out with either a constant amountof p4 and p6, or maintaining fixed the concen-tration of one of the proteins and varying the con-centration of the other protein. Figure 1B–D showthe nucleoprotein complex formed by both p4 and

p6. The results described above indicate that pro-tein p4 binds to sequence-specific sites separatedby 10 nt while protein p6 binding is not sequence-specific but binds at sites separated by 12 nt. Onthe basis of the footprint characteristics of eachprotein it seems that, in the presence of p6, proteinp4 maintains its contacts at positions 10 –30 betweenpromoters A2c and A2b (Figure 1B, lane e, and C,lanes a) and at positions 70 –120 between promotersA2b and A3 (Figure 1D, lane e, and E, lane g).However, there is an enlargement of the protec-tions 30 and 70 (Figure 1C and E), suggesting closeproximity between adjacent p4 and p6 moleculesor the stabilization of protein p4 upon p6 binding.There are five sets of protections with 12 bp spacerslocated between the p4 protections 30 and 70, corre-sponding to protein p6 binding (Figure 1C, lane a;D, lane e, and E, lane g). In addition, protein p6occupies the sequence from p4 position 10 to down-stream of promoter A2c (Figure 1B, lanes d and e).When increasing amounts of p6 were added, thefootprint of p4 at position 40 is modified first,suggesting that p6 occupies this area, and bindingtowards position 70 follows (Figure 1D, lanes c–e).Figure 2C shows a scheme of the binding ofproteins p4 and p6 obtained from data of severalexperiments.

Interplay between RNAP and the p4–p6binding at promoters A2c, A2b and A3

Ternary complex formation at early promoters A2cand A2b

At many E. coli promoters, the C-terminaldomain of the RNAP a subunit (aCTD) interactswith one or more A/T-rich sequences locatedupstream of the 235 element.14,15 Little is knownabout binding position of the sA-RNAP a subunitsat the B. subtilis promoters. To characterize theB. subtilis aCTD binding at promoters A2c andA2b, DNase I footprinting was carried out withmutant RNAP carrying a 59 amino acid residuedeletion at the carboxyl terminus of its asubunits.16 By homology with the a subunit ofE. coli, this deletion includes almost the entireaCTD domain. Comparison of the A2c promotersequence protected from DNase I digestion bywild-type RNAP (RPwt) and by the mutant RNAP(RP59) indicated that the aCTD domains arebound to the lower strand from positions 245 to259 and to the upper strand from positions 239to 257 relative to the promoter start site (Figure3A and B). In addition, a decrease in protection ofthe promoter sequence and in the hypersensitivitylocated at position 237 by the mutant enzymewas observed (Figure 3B), indicating that themutation impairs transcription complex stability.Further analysis of the transcription complexformed at promoter A2c by hydroxyl radical foot-printing (Figure 4) indicated that RNAP bindingresults in several protections downstream of thetranscription start site and in the 210 element

Transcriptional Regulators and Promoters Switch 359

Figure 2. A representation of protections by protein p4 (A), protein p6 (B) and the nucleoprotein complex p4/p6 (C) obtained by hydroxyl radical footprinting at the DNAsequence including promoters A2c, A2b and A3. The 210 and 235 elements for B. subtilis sA-RNAP at each promoter are indicated and the nucleotides protected by the pro-

(lanes d and e), and protection of a set of bandsaround positions 220, 230, 240, 250 and 260.Taken together, the results of hydroxyl radical andDNase I footprinting allows us to delimit the bind-ing of the aCTDs to the promoter A2c lower strandto sites centred at positions 241 and 251 relativeto the promoter start site. Analysis of RNAP bind-ing in the presence of p4/p6 reveals displacement

of the p4/p6 complex between positions 225 toþ10 (Figure 4, compare lanes i and j).

Binding of RNAP to promoter A2b is difficult todetect by footprinting, especially when the frag-ment includes promoters A2c and A3 and 100 mMsalt is used (Figures 3C and 5). It is known thatA2b is a strong promoter where RNAP formsunstable open complexes;17 therefore, this promo-ter is most probably not optimized for tight andstable RNAP binding. Figure 3C shows a compara-tive analysis of the DNase I footprinting obtainedwith the RNAP wild-type and the RNAP with theaCTDs deleted. The presence of a characteristichypersensitivity at position 241 and the weak

teins are marked by bars at both DNA strands. A includes p4 protections named 10 –120 here, comprising p4 bindingsites 1–4. Arrows indicate the inverted repeats at each binding site. B, The nucleotides protected by protein p6 withthe distances between them. C, The nucleotides protected by the p4/p6 complex when both protein p4 and p6 werepresent. Broken lines indicate the nucleotides protected in DNase I footprinting assays and arrowheads indicate hyper-sensitive positions.10 To facilitate the interpretation of the p4/p6 complex, we used ovals to represent protein p4 andcylinders to represent protein p6. In A, the G residues whose methylation interfered with protein p4 binding are inbold.12,13

Figure 3. DNase I footprinting of aCTD domain bind-ing to promoters A2c and A2b. Wild-type RNAP (RPwt)or mutant RNAP with the a subunits carrying a 59amino acid residue deletion at the C terminus (RP59)were used. A, The fragment contains only the sequencecorresponding to promoter A2c and was labelled withKlenow and [a-32P]ATP at an Eco RI digestion site. B,The fragment contains the sequence from position 4930p

to 5069, which includes promoters A2c and A2b andwas labelled with [g-32P]ATP at the 50 end at position4930 before PCR amplification. C, The fragment containsthe sequence from position 5165p to 4981, which includespromoters A2c and A2b, and was labelled with[g-32P]ATP at the 50 end at position 5165 before PCRamplification. Sequences protected by the a subunits atthe lower strand (footprint A) and upper strand (foot-prints B and C) are indicated by a in vertical bars. Dis-tances from promoter transcription start sites and thestart site position of promoters A2c and A2b are indi-cated in bp.

Figure 4. RNAP binding to promoter A2c in theabsence or in the presence of the p4/p6 nucleoproteincomplex analysed by hydroxyl radical footprinting.Binding of RNAP to promoter A2c was studied by add-ing increasing amounts of the enzyme (lanes b–e). Theeffect of the p4/p6 complex (lanes f–i) on RNAP bindingwas studied in the presence of 300 nM p4 and 12.5 mMp6. Controls with only protein p4 (lane k) or protein p6(lane l) or both p4 and p6 (lane j) were carried in parallel.The DNA fragment includes from nucleotide 4930 tonucleotide 5218 and was labelled with [g-32P]ATP at thetemplate strand of promoter A2c. The RNAP-bindingsite is depicted at the left. Several positions relative tothe promoter start site and protein p4 protections 10 –60

are indicated to the right. Binding locations for aCTD(a), and several regions protected by the RNAP (verticalbars) are denoted.


protection of a band at position 252 indicate bind-ing of the wild-type enzyme. Deletion of the aCTDdomains affects RNAP binding to this promoter,since the hypersensitivity at position 241 is greatlydiminished (Figure 3C). Binding of RNAP at pro-moter A2b analysed by hydroxyl radical footprint-ing shows slight protection from the 210 elementto position 253, with clear protected bands centredat position 251 (Figure 5). The latter should corre-spond to aCTD binding since it agrees with theprotection of the 252 band in the DNase I footprint(Figure 3C). Hence, at promoter A2b the aCTD hasthe strongest affinity to a site centred at position251 that corresponds to the distal part of an AT-rich sequence homologous to the UP elementdescribed for the rrnB P1 promoter.14 A second,lower-affinity binding site, which corresponds tothe proximal subsite of an UP element, can be dis-cerned at position 241 (Figure 5, left).

We analysed the role of the aCTD domains at thetranscription of promoters A2c and A2b in run-offexperiments with the wild-type RNAP or the

mutant RNAP aD59 (Figure 6). The results showthat deletion of the aCTDs led to a decrease of thetranscripts produced from promoters A2c andA2b by 68% and 96%, respectively; hence, theaCTDs are essential for transcription from promo-ter A2b and are required for full expression frompromoter A2c. In contrast, the aCTDs are notrequired for transcription from the viral constitu-tive A1 promoter (see Figure 6).

Ternary complex formed at late promoter A3

RNAP requires protein p4 to recognize promoterA3 efficiently due to the absence of a 235 element.To identify the binding sites of the aCTDs at pro-moter A3, we compared the DNase I footprints ofwild-type and truncated aD59-RNAP in the pre-sence of p4. As shown in Figure 7A, lanes c and e,RNAP is bound downstream of protein p4 onlywhen the wild-type aCTD domain was present.Comparison of the footprints of the wild-typeRNAP with those of the aD59-RNAP, in the pre-sence of p4, indicates that the aCTDs are boundimmediately downstream of p4, one at about pos-ition 249 and the other at about position 240relative to promoter A3 start site. The footprintsobtained when p4 was assayed with only theaCTDs, wild-type or D59, agree with the aboveconclusion (Figure 7A, lanes g–i). It is interestingto notice that hypersensitivities at positions 266and 276, which are induced upon p4 binding,became stronger only when wild-type RNAP wasadded, suggesting structuring of the promotersequence due to transcription complex formation.Hydroxyl radical footprinting of RNAP in the pre-sence of p4 (Figure 7B) shows protections at the210 element, at position 220, and sets of nucleo-tides centred at position 230. In addition, sets ofthree nucleotides centred at positions 242, 252,262, 273, 283 and 294 were protected. The latterset of protections matches the footprint obtainedwith p4 at positions 120 –70. From the DNase Iresults, the protections centred at positions 242and 252 should correspond to aCTD binding.The protection at position 262 could be originatedfrom the DNA bend due to p4 binding at site 3(protections 273, 283 and 294) which allowsp4–aCTD interaction. Therefore, aCTD displacesprotein p4 from site 4. Interestingly, the additionalpresence of protein p6 does not modify thefootprint at promoter A3 (Figure 7B, lane f).

Effect of the p4–p6 complex on open complexformation at promoters A2c, A2b and A3

In vitro, RNAP is capable of forming closed com-plexes at promoter A2c in the presence of proteinsp4 and p6, but formation of open complexes andthe synthesis of short transcripts are impeded.10

However, the latter results were obtained using aDNA fragment containing only promoter A2c andp4 binding sites 1 and 2. On the other hand,nothing is known about the step of transcription

Figure 5. Hydroxyl radical footprinting assay of thecomplex formed by the RNAP at promoter A2b in theabsence (lanes b and c) or in the presence (lanes d ande) of the p4/p6 nucleoprotein complex. Binding ofRNAP to promoter A2b was studied at two concen-trations of the enzyme (lanes b and c) and the effect ofthe p4/p6 complex (lanes d and e) on RNAP bindingwas carried out at the same concentrations of RNAP butin the presence of 300 nM p4 and 12.5 mM p6. Controlswith only protein p4 (lane h) or protein p6 (lane i), orboth p4 and p6 (lane g) were carried in parallel. TheDNA fragment used includes from nucleotide 5164 tonucleotide 4930 and was labelled with [g-32P]ATP at thecomplementary strand. The location of the 210 promo-ter element and putative a subunits binding are depictedto the left. Protein p4 protections 70 –100 and their pos-ition relative to the promoter start site are depicted tothe right. The panel to the left shows an expansion ofpromoter A2b from positions 255 to about þ9.


at promoters A2b and A3 affected by the p4/p6multimeric complex. Since the entire p4/p6 com-plex could be required for efficient repression andsimultaneous switch of the promoters, we studiedits effect on open complex formation at promotersA2c, A2b and A3 with a DNA fragment encom-passing positions þ77 to þ68 relative to promoters

A2c and A3 start sites, respectively. To that end, weallowed open complex formation in the presence ofp4 and in the absence or presence of protein p6.The results, shown in Figure 8, indicated thatopen complex was formed faster at promoter A3than at promoter A2c when only p4 was added(lanes c and d). The additional presence of p6 (i.e.

Figure 6. In vitro transcription from promoters A2c and A2b depends on the presence of the aCTD domain. Run-offassays were performed with a mixture of fragments, one including promoters A2b and A2c and the other with onlypromoter A1. Transcription from these fragments gives rise to a 30 nucleotide transcript from promoter A1 or 77 and172 nucleotides when driven from promoters A2c and A2b, respectively. RNAP wild-type (RPwt) or with truncated asubunits (RP59) were added at the concentrations indicated.

Figure 7. aCTD domain bindingat promoter A3. A, DNase I foot-printing analysis of the bindingpositions of the B. subtilis sA-RNAPa subunits at promoter A3, in thepresence of protein p4. The proteinswere added as indicated above theautoradiograms. Wild-type RNAP(RPwt) or RNAP with the a sub-units carrying a 59 amino acid resi-due deletion at the C terminus(RP59), as well as the correspond-ing purified a subunits (awt anda59) were used. Protections due tothe RNAP and to the a subunitsare indicated. The distance fromthe A3 promoter transcription startsite is indicated in bp. B, Hydroxylradical footprinting of the transcrip-tion complex formed by the RNAPat promoter A3 in the presence ofp4/p6 nucleoprotein complex. Theeffect of protein p6 on the transcrip-tion complex formed in the pre-sence of protein p4 was assayed byusing 25 nM RNAP, 300 nM proteinp4 and 12.5 mM protein p6. Con-trols with only RNAP, only p4 orp6, or a mixture of proteins p4 andp6 were carried in parallel. The

DNA fragment includes from nucleotide 5218 to nucleotide 4930 and was labelled with [g-32P]ATP at the lower strand.Protein p4 protections 70 –120 are depicted at the left. Several positions relative to the promoter start site are depicted atthe right. Some protected nucleotides upon RNAP binding are indicated on the autoradiogram.


the multimeric p4/p6 complex) stabilised promo-ter A3 open complex while destabilising promoterA2c open complex (lanes h–l). At promoter A2c,open complex was formed faster when both pro-teins, p4 and p6, were added (compare lanes cand h), probably due to the p6-mediated stabiliz-ation of p4 at site 1, which should increase therecruitment of the RNAP to the promoter.12 At pro-moter A3, the transition from preformed closedcomplex to open complex was 2.7 times fasterwhen p6 was added in addition to p4 (data notshown). Therefore, p6 seems not to be required forformation of the transcription complex at promoterA3, and its role in the activation of transcriptionmust be subsequent to closed complex formationby stimulating events leading to productive opencomplex. In this and other experiments, we couldnot detect any open complex at promoter A2b.

Discussion

Position of proteins p4 and p6 in the DNAsequence enclosing promoters A2c, A2band A3

Transcripts produced in vivo from promoters A2c

and A2b are synthesized soon after infection,reaching a steady-state level at 20 minutes postinfection, while promoter A3 expression starts atabout minute 15.18,19 Analysis of the half-life ofthese transcripts indicates that promoters A2c andA2b are repressed only partially by the synthesisof proteins p4 and p6. In vitro, protein p4 contactstwo regions (named here 10 –60 and 70 –120) locatedwithin the 219 bp that separate the promoters A2cand A3 start sites. Each region has six p4 sets ofprotections located about 10 bp apart, thus on thesame DNA face, and four helix-turns separate oneregion of six from the other (Figure 2A). Protec-tions at positions 10 –60 encompass sites 1 and 2,and here we describe for the first time site 2 as abona fide binding site for protein p4. Protections atpositions 70 –90 are located at p4 binding site 3,while protections 100 –120 define a new p4-bindingsite, site 4. Thus, in the sequence from promoterA2c to promoter A3 there are four p4 bindingsites, 1–4, although p4 affinity differs, site 4 beingthe binding site with lower affinity. It has beenshown that protein p4 is a dimer in solution, andprevious stoichiometric titrations indicate that twop4 dimers could occupy the sequence containingbinding site 3.20 However, since in these exper-iments the sequence analysed included also totallyor partially site 4, the possibility that the dimer isthe p4 unit bound to each binding site cannot beruled out. Therefore, two or four p4 dimers couldbe located between the co-oriented promoters A2cand A2b and another two or four between theoppositely oriented promoters A2b and A3 (seeFigure 2A). Binding of p4 to a fragment encom-passing sites 3 and 4 induces strong curvature ofthe sequence,21 and we have recently found thatp4 induces fairly the same bent angle to thesequence containing sites 1 and 2 (L. Perez, M.S. &A.C., unpublished results). On the other hand,methylation interference assays indicated thatguanine residues whose methylation interferedwith protein p4 binding were located at theinverted repeat on sites 1 and 3 (Figure 2A).12,13

These data suggest that central protections oneach binding site (protections 20, 50, 80 and 100)could arise from a protein p4-induced local bendof the DNA. The organization of bound p4molecules together with the presence of hydroxylradical protections at the middle of the bound p4proteins between protections 60 and 70 (Figure 1C,lane b), suggests that the complete occupation ofthe p4 binding sites could twist the DNA helix togive rise to a higher-order structure, as is the casefor the loop induced at the E. coli gal promotersequence by GalR repressor.22

Protein p6 contacts, at specific positions, almostevery 12 bp on both DNA strands of the fragmentunder study, and several of the p6 protectionsoverlap the p4 binding sites. From the results pre-sented here (see Figure 2C), p4 occupies sites 1, 3and 4, and p6 binds downstream of promoter A2cin the p4/p6 complex. However, the set of p6 pro-tections located between p4 bound to sites 1 and

Figure 8. Open complex formation at promoters A2cand A3 in the presence of protein p4 or proteins p4 andp6. Open complex formation at promoters A2c and A3was analysed by potassium permanganate footprinting.Proteins were added as indicated above the autoradio-gram and the open complex formed was analysed at thetimes indicated. RNAP was at 25 nM, protein p4 was at300 nM and protein p6 was at 12.5 mM. Promoters A2cand A3 are indicated.


3, although spaced by 12 bp, differ in position fromthe pattern obtained with only p6. This new pat-tern could arise from protein-induced structure inthe DNA or by the formation of a nucleosome-likestructure through the relocation of protein p6orchestrated by protein p4 (Figure 2C). Since pre-vious DNase I analysis of the p4/p6 complexreveals almost complete protection of the DNAsequence between p4 sites 1 and 3 (see Figure2C),10 we assume that in the p4/p6 complex theDNA is wrapped around a core of proteins p4 andp6. This would resemble the nucleosome-likestructure proposed for binding of TraM to its cog-nate site at oriT.23 The enlargement of p4 protectionat positions 30 and 70 in the presence of p6 mostprobably reflects stabilization of p4 at bindingsites 1 and 3, in agreement with the demonstratedreciprocal synergy of p6 on p4 binding.10 Themechanism for the synergy between p4 and p6 dif-fers from those described for other transcriptionalregulatory proteins. Protein IHF bends the DNAto facilitate regulator–RNAP interactions.24 In con-trast, at the f29 promoters A2c and A3 there arep4–RNAP interactions in the absence of proteinp6.21 On the other hand, at promoter nrfA, repres-sors FIS and IHF are removed from the promoterby the activator proteins NarL and NarP,25 whileat promoter melAB, CRP stabilizes binding ofMelR at a low-affinity binding site located justupstream of the promoter, resulting in MelR andRNAP sigma subunit contacts.26 In our case, thedata indicate that the regulator protein p4 reposi-tions histone-like protein p6 to control promotersA2c, A2b and A3. A loop structure resulting fromthe interaction between p4 bound at sites 1 and 3may be required for accuracy in the regulation ofthe promoters; however, loop formation is energe-tically unfavored by the 100 nucleotides distance.The free energy needed to deform the DNA helixcould be provided by the adjacent p6 boundmolecules.

Binding of RNAP to promoters A2c, A2b and A3

In E. coli RNAP, the aCTD domain interacts withA/T-rich sequences upstream of the 235 elementresembling the UP elements found at the rrn pro-moters, and functions primarily by increasing theinitial binding of the RNAP14,15,27,28 Here, wedemonstrate that the B. subtilis aCTDs are requiredfor transcription complex stabilization at promotersA2b and A2c, since RNAP–promoter interactionwas impaired strongly when the 59 carboxyl-term-inal amino acid residues were deleted. Thus, asdescribed for promoter rrnB P1, the DNA-bindingsites for B. subtilis RNAP-aCTD constitute a thirdrecognition element at promoters A2c and A2b.Our analysis reveals two binding sites for theaCTD domains at promoter A2c centred at pos-itions 241 and 251 relative to the promoter startsite. At promoter A2b, within an intrinsicallycurved sequence,21 aCTD binds preferentially tothe distal half of the A/T-rich region homologous

to the UP element and present between positions240 and 254 from the promoter start site (seeFigure 2). In addition, we show that in the presenceof protein p4, aCTD binds at promoter A3 at pos-itions similar to those at promoter A2c (positions240 and 250) even though promoter A3 does nothave a detectable UP element. Since p4 interactswith aCTD and both proteins are placed twohelix-turns apart, the bend of the DNA producedby p4 binding will allow this interaction. All thesedata lead us to conclude that, irrespective ofwhether UP elements are present, the positioningof the B. subtilis aCTD in the promoters underanalysis seems to be dictated principally by thebinding of RNAP.

In the p4/p6 complex, p4 occupies positionscentred at positions 230, 240 and 250 from thestart sites of promoters A2c and A2b, implyingthat there is overlap with the aCTD and with the235 elements (see Figure 9). This organizationwould place the bound p4 dimers equidistantwith respect to promoters A2c and A2b start sites,and one helix turn downstream from position þ1of late promoter A3 (see Figure 2A). It is temptingto speculate that these differences could be criticalfor stable RNAP–promoter interaction.

In summary, repression of early transcriptionfrom promoters A2c and A2b relies on the syner-getic binding of two regulators to the promotersequence resulting in a new complex able toantagonize the binding of a higher binding affinityprotein, the RNAP, through competition with thesame target DNA. Activation of the late promoterA3 takes place mainly by the stabilization of theRNAP through its interaction with the p4/p6nucleoprotein complex.

Control of promoters A2c and A2b by feed-back regulation

We envision the switch from early to late tran-scription as the interplay between the RNAP andthe p4/p6 complex for binding to the sequencecontaining promoters A2c, A2b and A3. AlthoughRNAP would have the higher binding affinity,efficient complex formation and transcriptioninitiation requires appropriate positioning. At thispoint, the relative location and stability of p4 bind-ing with respect to promoters A2c and A2b versuspromoter A3 could be critical. At the onset of infec-tion, there are few molecules of p4 and p6; there-fore, RNAP recognizes and transcribes promotersA2c and A2b, but not promoter A3. As the concen-tration of proteins p4 and p6 (expressed frompromoters A2c and A2b) increases, a p4/p6nucleoprotein complex extends over the region,interfering with aCTD binding to promoters A2cand A2b and most probably impairing the correctinteraction of the RNAP s subunit with the 235element. Therefore, the efficiency of transcriptioncomplex formation at promoters A2b and A2cdecreases, accounting for the steady state of theearly transcripts reached 20 minutes after infection.


However, if promoters A2c and A2b were totallyrepressed at a time when exponential DNA syn-thesis occurs, synthesis of the early proteinsexpressed from these promoters, and required forreplication, could be compromised. The phagecould overcome this situation by maintaining acontrolled synthesis of early proteins by a feed-back regulatory mechanism.

Materials and Methods

Proteins and nucleotides

B. subtilis RNAP was purified as described.29 Therecombinant RNAP wild-type and the RNAP with the59 amino acid residue deletion at the carboxy terminusof the a subunits were purified as described.16 Proteinsp6 and p4 were purified as described.30,31 UnlabelledNTPs and dNTPs were purchased from Pharmacia.[g-32P]ATP (3000 Ci/mmol), [a-32P]UTP and [a-32P]dATP(3000 Ci/mmol) were purchased from Amersham Inter-national.

DNA substrates

The DNA fragments used in most of the experiments,except for the one used in Figure 3 (indicated in theFigure legend), were obtained by PCR amplificationfrom full-length f29 DNA with synthetic primers. Oneof the primers was labelled by treatment with polynu-cleotide kinase and [g-32P]ATP for 45 minutes at 37 8Cprior to amplification. Each fragment was purified byNuSieve GTG agarose (FMC) gel electrophoresis andQuiagen gel extraction kit.

Hydroxyl radical footprinting

End-labelled promoter-containing fragments wereincubated at 20 8C with the indicated proteins in100 mM KCl, 10 mM MgCl2, 25 mM Tris–HCl (pH 7.5),1 mM EDTA and 1 mg of bovine serum albumin (BSA)in a final volume of 50 ml. Proteins p4 and p6 were incu-bated for five minutes at 0 8C prior to the addition ofthe RNAP and then incubation proceeded for 20 minutesat 20 8C. Hydroxyl radical cleavage was carried out asdescribed.32,33 Digestion products were analysed by elec-trophoresis on 6% polyacrylamide gels in the presenceof 8 M urea.

DNase I and permanganate footprinting

Footprint reactions contained end-labelled DNA,

100 mM KCl, 10 mM MgCl2, 25 mM Tris–HCl (pH 7.5),1 mg of poly[d(I 2 C)], 1 mM EDTA and 1 mg of BSA ina final volume of 20 ml. Proteins p4 and/or RNAP wereadded in the amounts indicated and incubated for 20minutes at 37 8C. DNase I footprinting was performedwith 0.05 unit of RQ1-DNase I (Promega) at 37 8C fortwo minutes.34 Reactions were stopped by the additionof EDTA (to 10 mM) and 10 mg of tRNA. For KMnO4

footprinting, the reagent was added up to 4 mM, incu-bated for 30 seconds, and then stopped with 1 M b-mer-captoethanol. DNA was ethanol-precipitated and thencleaved with 1 M piperidine following the standardDNA sequencing procedure.35 For each footprintingexperiment, DNA was precipitated with ethanol andanalysed on denaturing 6% polyacrylamide gels.

In vitro transcription assay

Run-off transcription assays (25 ml) contained 25 mMTris–HCl (pH 7.5), 10 mM MgCl2, 2 mM ditiothreitol,2 mg of poly[d(I 2 C)], 100 mM KCl, 10 units of RNasin,100 mM each GTP, ATP and CTP, 50 mM [a-32P]UTP(1 mCi), 200 mM GpU and 2 nM DNA template. Thetemplate was a mixture of two fragments, one containingpromoters A2c, A2b and A3 and the other containingpromoter A1. RNAP was added and, after 20 minutes at37 8C, reactions were stopped by addition of SDS to0.15% (w/v) and EDTA (to 2.5 mM). Transcripts wereanalysed by electrophoresis in denaturing 20% poly-acrylamide gels. Quantification of the transcripts wascarried out by using a Fuji Bas-IIIs Image analyser.

Acknowledgements

We thank L. Rothman-Denes and F. Rojo forstimulating discussions and critical reading of thismanuscript. We are indebted to J. M. Lazaro andL. Villar for protein purification, and to W. Rossfor kindly supplying the hydroxyl radical foot-printing protocol. This work was funded by grants2 R01 GM27242-24 from the National Institutes ofHealth and BMC 2002-03818 from the SpanishMinistry of Science and Technology and by anInstitutional grant from Fundacion Ramon Arecesto the Centro de Biologıa Molecular “SeveroOchoa”. The “Ramon y Cajal” program of theSpanish Ministry of Science and Technologysupported A.C.

Figure 9. A representation of RNAP and proteins p4 and p6 binding sites defined in this study. Broken lines indicatethe nucleotides protected in DNase I footprinting assays and arrowheads indicate hypersensitive positions. Ovals rep-resent protein p4 and cylinders represent protein p6. RNAP is represented at the top of the scheme, with a and s sub-unit binding locations indicated.


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Edited by I. B. Holland

(Received 2 October 2003; received in revised form 1 December 2003; accepted 9 December 2003)


molecular interplay between rna polymerase and two transcriptional regulators in promoter switch

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