DNA microloops and microdomains: a general mechanism for transcription activation by torsional...

Article No. mb981834 J. Mol. Biol. (1998) 279, 1027±1043

REVIEW

DNA Microloops and Microdomains: A GeneralMechanism for Transcription Activation byTorsional Transmission

Andrew Travers1* and Georgi Muskhelishvili2*

1MRC Laboratory of MolecularBiology, Hills Road, CambridgeCB2 2QH, England2Institut fuÈ r Genetik undMikrobiologie, LMU MuÈ nchenMaria-Ward-Straûe 1a80638 MuÈnchen, Germany

Abbreviations used: UAS, upstresequence.

0022±2836/98/251027±17 $30.00/0

Prokaryotic transcriptional activation often involves the formation ofDNA microloops upstream of the polymerase binding site. There issubstantial evidence that these microloops function to bring activator andpolymerase into close spatial proximity. However additional functionsare suggested by the ability of certain activators, of which FIS is the bestcharacterised example, to facilitate polymerase binding, promoter open-ing and polymerase escape. We review here the evidence for the conceptthat the topology of the microloop formed by such activators is tightlycoupled to the structural transitions in DNA mediated by RNA polymer-ase. In this process, which we term torsional transmission, a majorfunction of the activator is to act as a local topological homeostat. Weargue that the same mechanism may also be employed in site-speci®cDNA inversion.

# 1998 Academic Press

*Corresponding authors

Introduction

DNA untwisting is a crucial step in recombina-tion and in the initiation of transcription and DNAreplication. At the simplest level this can beachieved by the binding of a protein dimer whichthen acts as a torque-wrench untwisting the DNAbetween the half-sites (Figure 1A). A good exampleis provided by the MerR protein. Here untwistingis dependent on the binding of an effector, Hg2�,which triggers a conformational change in the pro-tein (Ansari et al., 1992). Other examples includethe restriction enzyme EcoR1 (Lesser et al., 1993)and certain mutants of the Gin invertase (Klippelet al., 1993; Merker et al., 1993). In this simple scen-ario the protein on binding isolates a separatetopological DNA domain distinct from the ¯ankingregions. However the DNA topology of many ofthe protein assemblies that catalyse recombination,transcription initiation or replication initiation ismore complex and often involves sequential topo-logical transitions (Amouyal & Buc, 1987;Muskhelishvili et al., 1997).

It is well established that large nucleoproteincomplexes can constrain a distinct local DNA top-ology (Saucier & Wang, 1972) but the distribution

am activating

of this constraint within a complex is often illde®ned. We shall argue that not only does theDNA bound within the complex constitute a topo-logical entity largely independent of the ¯ankingDNA but also that functionally distinct topologicaldomains exist within the complex itself. Thesemicrodomains, which in principle can be as shortas one duplex turn as in the MerR-DNA complex,are delimited to a greater or lesser degree by pro-tein-DNA contacts within the complex and in somecases take the form of short DNA loops (micro-loops) of 50 to 100 bp in extent (Figure 1B). Theseparation of different topological domains withinan enzymatically active complex allows the possi-bility that the protein components could mediatetopological coupling, or torsional transmission,between the domains. Here we review the evi-dence for this concept and note that this mode oftopological coupling is distinct from that describedby Bowater et al. (1994) in which the diffusion ofsuperhelical tension generated by the processiveprogression of RNA polymerase directly affects theactivity of a nearby promoter by altering the super-helical density of a relatively large DNA domain.By contrast, topological coupling mediated by dis-tinct microdomains within an enzymatically activelarge complex allows the torsional strain to bereleased in discrete steps and used for overridingsubsequent kinetic barriers in the reaction. This

# 1998 Academic Press

Figure 1. DNA microloops andmicrodomains. A, Untwisting ofDNA by a dimeric DNA-bindingprotein. The region between theprotein contacts constitutes thetopologically isolated DNA micro-domain. B, Reversible transitions inDNA microloops. The Figure showshow a microloop can adopt alterna-tive con®gurations as an untwistedplanar curve or a left-handedwrithe. The handedness of thewrithe depends on the helicalrepeat between the midpoints ofthe protein dimer binding sites.

1028 Review: Transcription Activation

process is thus formally analogous to the stepwiserelease of energy in catabolic enzymatic reactions.

Prokaryotic transcription initiation

The evidence for torsional transmission isderived principally from studies of transcriptioninitiation at prokaryotic stable RNA promoters(Muskhelishvili & Travers, 1997). However theparadigm for the dissection of the initiation processis the lac promoter (Figure 2A). Several distinctphases in the pathway of transcription initiation atthis promoter by the s70 holoenzyme have beendistinguished (Buc & McClure, 1985). An initialrapid recognition of the ÿ35 region is characterisedby an increased sensitivity of the s subunit to pro-teolytic digestion without any evidence for anextended interaction downstream of position ÿ25.This stage is followed by the formation of an inter-mediate complex in which the polymerase ``foot-print'' extends downstream to position � �20 andthe DNA within the ÿ10 region becomes sensitiveto chemical reagents which target untwisted DNA(Spassky et al., 1985). In this con®guration the ssubunit makes direct close contacts to the ÿ10 andÿ35 hexamers (M. Jacquet, H. Buc and M. Buckle,personal communication). The next step is the cre-ation of a single-stranded region around the tran-scription startpoint which allows the non-codingstrand to serve as a template for transcription.Following transcription initiation the breaking ofs-DNA contacts is necessary for the ®nal escape ofpolymerase from the promoter region.

An essential requirement for topological tran-sitions is that the DNA within individual microdo-

mains can be readily distorted, either by bendingor untwisting or both. This implies the microdo-mains should contain DNA sequences selected forthis property. In s70 dependent promoters suchselection is apparent. The region containing andupstream of the ÿ35 hexamer can either form acoherent intrinsic bend (Plaskon & Wartell, 1987)or is anisotropically bendable in the same direction(Travers, 1991). However downstream of the ÿ35region there is a change in direction of potentialbending such that the downstream bend is rotatedby 2±3 bp corresponding to an angle of �70 to100� with respect to the upstream bend (Figure 2B).Another general characteristic of promoters is thatthe DNA sequences in the central region of the ÿ35to ÿ10 spacer are, on average, more ¯exible incomparison to the short sequences in nucleosomalDNA which determine anisotropic ¯exibility(Travers, 1991).

In the initiation process one step at which tor-sion is generated is the transition from the initial tothe intermediate complex (see Figure 2A). This stepis known to possess a high �Cp, indicative of a sig-ni®cant conformational change in the protein (Roeet al., 1985), and consistent with the extension ofthe polymerase footprint downstream of the start-point. We suggest that the extension of the poly-merase contact downstream of the startpointestablishes a microdomain encompassing the ÿ10region and the startpoint and distinct from the ÿ35to ÿ10 region (Figure 2C). The resultant generationof torsion must be assumed to require a rotation ofthe DNA or protein or both. This could be accom-plished by a relative rotation of the microdomainscontaining the upstream and downstream bends toform a coherent or near-coherent bend (Figure 2B).

Figure 2. A, Stepwise scheme for the initiation of transcription by E. coli RNA polymerase. B, Postulated change inDNA con®guration on transition from the initial to the intermediate complex. C, Establishment of DNA microdo-mains in the intermediate initiation complex. The indicated microdomain upstream of the ÿ35 region would bedelimited in some promoters by contacts in the ÿ35 region and the UP element. D, Microloop formation by RNApolymerase by upstream DNA contacting the backside of the enzyme. E, Stabilisation of a microloop by a DNA-bending protein. FIS is shown as an example.

Review: Transcription Activation 1029

This motion would produce signi®cant DNAunwinding within the polymerase-DNA complexand could be suf®cient to nucleate untwistingwithin the ÿ10 region at the less stable TA basesteps (Patel et al., 1983; Drew et al., 1985) and/orwithin the ¯exible region in the ÿ35 to ÿ10 spacer.The former mode of untwisting could be directlytrapped by interaction with the helical 2.3 regionof the s70 subunit (Malhotra et al., 1996). Sub-sequent promoter opening requires extension ofthe untwisted region to the transcription startpoint.Topologically this motion may be regarded as ana-logous to the progression of polymerase duringchain elongation (Liu & Wang, 1987) such that theforward translocation of the untwisted region iscoupled to negative torsion behind the bubble. Wepropose that this motion again involves a rotationof the leading end of the polymerase relative to thetrailing end (Figure 2C) and is thus also formallycompatible with the ``inchworm'' and ``movingdomain'' models for transcription elongation(reviewed by Heumann et al., 1997). Whatever theprecise mechanism, the topological transitionsduring the nucleation process should be distin-guished from those operating during promoteropening and polymerase escape.

The concept of microdomains requires the div-ision of the overall topological constraint in anucleoprotein complex between two or more topo-logically separable entities. At lacP the topologicalconstraint associated with initiation complex for-mation on closed circular DNA does not parallelDNA untwisting and strand separation (Amouyal& Buc, 1987). The approximate linking numberchanges (which quantify the extent of DNA distor-tion) associated with initial, intermediate and opencomplex formation are ÿ1.4, ÿ1.2 and ÿ1.8,respectively. This result implies, as the authorssuggested, some conversion of negative writhe tountwisting. A possible explanation for the con-straint of negative superhelicity in the initial com-plex is the formation of a microloop withsequences upstream making an additional contactwith RNA polymerase (Figure 2D). The existenceof such loops has been inferred both from theenhancement of promoter activity by upstreamcurved DNA at lac and other promoters (Braccoet al., 1989; Gartenberg & Crothers, 1991; Ellingeret al., 1994b) and also from the activation of the lpL and malT promoters by the DNA bending pro-tein IHF (Giladi et al., 1990; DeÂthiollaz et al., 1996;see also extensive reviews by PeÂrez-MartõÂn et al.,1994 and Rippe et al., 1995). More direct evidence


for an upstream polymerase contact around pos-itions ÿ85 to ÿ90 at the lac UV5 promoter has alsobeen presented (Buckle et al., 1992). Not only isthere evidence that the upstream region of lacP canform a microloop, but both with and without theDNA-bending protein CRP Zinkel & Crothers(1991) observed changes in the overall direction ofcurvature of DNA entering and exiting theinitiation complex during the transition from theintermediate to the open complex. This they inter-preted as a change in the topology of the micro-loop. Although the functional signi®cance ofmicroloop formation and its associated topologicalchanges at this promoter remain to be established,the microloop itself could constitute an additionalmicrodomain delimited by simultaneous contactsof polymerase with the promoter recognitionelements and with upstream DNA.

A further aspect of promoter function is thenecessity, in some cases, to achieve optimal rates ofinitiation. This requires on the one hand that theaf®nity of the polymerase for the promoter sitemust be suf®ciently high to ensure optimal promo-ter opening. On the other hand high af®nity bind-ing may be deleterious to promoter clearance(Ellinger et al., 1994a). These opposing require-ments can be addressed by different facets ofpromoter design which both permit tight bindingand facilitate clearance.

Stable RNA promoters

The stable RNA promoters are, as a class, amongthe most active promoters in Escherichia coli. Yetthe core promoter region normally contains at leastthree deviations from the consensus structure(Lamond & Travers, 1985a). The ÿ35 hexamer is

Figure 3. The organisation of the tyrT promoter. This orgaindicates the transcription startpoint; G

C , the G�C-rich discrimerase binding; S, the 16 bp spacer between the hexamers; I

often suboptimal with respect to the ``consensus''sequence while many of the strongest promotershave a suboptimal spacer of 16 bp (Figure 3). Inaddition most of these promoters contain a G�C-rich region between the ÿ10 hexamer and the tran-scription startpoint (Travers, 1980a). This so-called``discriminator'' is believed to impose a kineticblock to the formation of the open complex byproviding a barrier for DNA untwisting. It alsoconfers sensitivity to the nucleotide ppGpp (Cashel& Gallant, 1969) in vitro (Travers, 1980b;Mizushima-Sugano & Kaziro, 1985; Lazarus &Travers, 1993) and to stringent control in vivo(Lamond & Travers, 1985b; Zacharias et al., 1989;Ninnemann et al., 1992; Josaitis et al., 1995). For themost active promoters in this class optimal activityrequires an upstream activating sequence (UAS,otherwise UAR and USE) extending to 130 to150 bp proximal to the startpoint (Lamond &Travers, 1983; Gourse et al., 1986; van Delft et al.,1987; Bauer et al., 1988). This region contains twodistinct activator elements, an UP element betweenÿ40 and ÿ60 (Ross et al., 1993) and binding sitesfor the DNA bending protein FIS further upstream.The UP element is a binding site for the a subunitsof RNA polymerase and acts in concert with theÿ35 region to recruit RNA polymerase to the pro-moter (Rao et al., 1994). In the stable RNA promo-ters so far characterised in detail (rrnB P1, tufB,tyrT) the FIS binding sites are conserved in numberand phase. In particular the phase of the three highaf®nity sites is maintained at a helical repeat of10.2±10.3 bp. The position of site I, closest to thetranscription startpoint, is the most highly con-served with a midpoint at position � ÿ71. The pos-itions of sites II and III are less conserved such thatthe separation of the midpoints of sites I and III

nisation is typical of stable RNA promoters in E. coli. �1minator; ÿ10 and ÿ35, the two hexamers directing poly-, II, III, FIS binding sites.


varies from 52 bp in tyrT to 73 bp in rrnB P1. Inaddition in both rrnB P1 and tyrT site II has a sub-stantially lower af®nity for FIS than sites I or III(Ross et al., 1990; Lazarus & Travers, 1993). How-ever inspection of the sequences of other stableRNA promoters indicates substantial variability inthe number and quality of FIS binding sites. Forexample, rrnE P1 apparently lacks a high af®nitysite at position � ÿ71 (Lazarus, 1992) whilepromoters directing tRNA, rather than rRNA,production show even more apparent variation. Inaddition to FIS binding sites the UAS regions ofboth the tyrT and rrnB P1 promoters contain bind-ing sites for another abundant DNA-architecturalprotein, H-NS (Tippner et al., 1994; L. Seeley &Travers, unpublished observations).

The UAS regions of stable RNA promoters canenhance transcription in two ways: factor-indepen-dent and factor-dependent (Newlands et al., 1991).Although it has been suggested that the UPelement is suf®cient for factor-independent acti-vation of the rrnB P1 promoter (Zacharias et al.,1992) at tyrTp an insertion mutation of 5 bp at pos-ition ÿ98 decreases the af®nity of the promoter forRNA polymerase by �tenfold, indicating thatsequences upstream of this position are requiredfor optimal promoter function (Muskhelishvili et al.,1997). One interpretation of this extended sequencerequirement is that, as for lacP, the tyrT UAS formsa microloop making an additional contact withRNA polymerase upstream of the 5 bp insertionpoint (Muskhelishvili et al., 1995, 1997). In someother s70-dependent promoters RNA polymerasealso induces phased DNase I hypersensitive sitesin the upstream region suggesting extendedupstream contacts (Landini & Volkert, 1995;Nickerson & Achberger, 1995).

Mechanism of transcriptionalactivation by FIS

What role do the multiple FIS binding sites playin transcription initiation at stable RNA promo-ters? There is substantial in vitro evidence that atboth the rrnB P1 and tyrT promoters FIS recruitsRNA polymerase into an initial complex and thusincreases KB (Bokal et al., 1995; Muskhelishvili et al.,1995, 1997). However whereas site I is apparentlysuf®cient in vitro for this process at rrnB P1, attyrT an insertion mutation of 5 bp which disruptsthe helical phasing of the FIS binding sites pre-vents the recruitment of polymerase to form theinitial FIS-polymerase-promoter ternary complex(Muskhelishvili et al., 1995). This provides strongevidence that the helical phasing of the FIS bindingsites is crucial for function as also observed in vivofor rrnB P1 (Zacharias et al., 1992). In addition FISactivates subsequent steps in the initiation path-way. At rrnD P1 FIS facilitates the transition to theelongating complex (Sander et al., 1993) while attyrT FIS stimulates both promoter opening andpolymerase escape (Muskhelishvili et al., 1997).

Importantly, activation at these latter two stepsagain requires that the FIS sites in the UAS be inhelical register. Thus FIS, uniquely for a prokaryo-tic transcriptional activator, is involved throughoutthe initiation process in vitro.

What is the molecular basis for the activation ofsequential steps by FIS? The coherent DNA bend-ing induced by FIS in the UAS could increase boththe probability of forming a microloop and its sub-sequent stability (Figure 2E). Such an effect wouldbe consistent with the inability of the �5 insertionmutant to support the formation of a FIS-polymer-ase-DNA complex (Muskhelishvili et al., 1995) or topromote FIS-dependent DNA untwisting in theÿ10 region (Muskhelishvili et al., 1997). Similarly,the FIS-dependence of post-initiation events at thewild-type tyrT promoter implies that the integrityof the loop is maintained during the initial stagesof transcription elongation. Mechanistically the roleof FIS in facilitating the initiation process could beexplained by assuming that FIS stabilises a left-handed writhe. In this model the writhed micro-loop captures the polymerase in the initial com-plex. Once formed this microloop constitutes adistinct topological domain. Subsequently RNApolymerase rotates by aligning the ÿ10 and ÿ35promoter elements during the formation of theintermediate complex. This rotation writhes theloop in a right-handed sense generating torsionwithin the microloop (Figure 4). FIS then drives areversion to left-handed writhe. This motion bothtransmits the torsion accumulated in the microloopas untwisting to the separate topological domainformed by the initiation bubble and accommodatesthe negative superhelicity generated upstream bythe movement of the elongation bubble (Figure 4).In this model torsional transmission could bemediated by direct FIS-polymerase contacts(Muskhelishvili et al., 1995), by polymerase con-tacts with UAS DNA or alternatively by both typesof contacts. An important attribute of the FIS-induced loop is that while maintaining itself as aseparate topological domain it is suf®ciently ¯ex-ible to accommodate a redistribution of twist andwrithe and so assume different topologicalcon®gurations.

The crucial step in this process is the transitionfrom the initial to the ``intermediate'' complex. Weenvisage that this step involves a rotation betweenthe two parts of the promoter that brings theupstream and downstream curves into approxi-mate register (Figure 2B). It is this rotation thatgenerates torsional strain and is the primary driv-ing force for promoter opening and polymeraseescape.

A critical experiment to test this model would beto place speci®c nicks or gaps between the proxi-mal FIS binding site and polymerase, and alsobetween the FIS binding sites. The model predictsthat such nicks should not greatly affect the initialbinding but should block the facilitating effects ofFIS on subsequent steps in the initiation reaction.

Figure 4. The torsional transmission model for transcriptional activation by FIS. The types of polymerase complexes,as well as the topological alterations in twist (Tw) and writhe (Wr) accompanying the transitions between the com-plexes, and rotation of polymerase (Rot) are indicated. The arrowheads indicate the direction in which the alterationsproceed. The polymerase is drawn as an ellipse, DNA is represented by a thin line. The red circles in the DNA loopindicate the accommodated torsion, the small dark-grey ellipse represents the initiation and elongation bubbles. FIS isomitted from the drawing for clarity. For further details see the text.


Role of DNA geometry

The importance of DNA geometry in a differentpromoter context has been inferred by Hirota &Ohyama (1995) who demonstrated a preference fora right-handed writhe at certain stages of theinitiation process. They showed that putative right-handed curves placed upstream of the ÿ35 hexam-er of the bla promoter were more effective thanplane curves in promoting initial complex for-mation and promoter opening. By contrast left-handed curves repressed transcription. In this con-

text we note that the eukaryotic UBF transcriptionfactor which binds immediately upstream of rRNApromoters wraps DNA as a positive supercoil(Putnam et al., 1994).

For tight topological coupling we anticipate thatmicroloops and other microdomains are suf®-ciently short to prevent dissipation of the storedtorsion. In large loops any stored torsion is dissi-pated by accommodation of a DNA conformationwith lower free energy, i.e. by inducing additionalwrithe. In practice the formation of additionalwrithe will be limited by the bending energy


necessary to form tight loops and consequently thelength of microloops is likely to be restricted to anupper limit of 100 to 150 bp. A further feature of aconstrained microdomain is that its geometry, atleast initially, is determined by the local helicalrepeat of the DNA separating the delimitingprotein binding sites (see legend to Figure 1). Forexample for FIS binding sites in the UAS regions ofstable RNA promoters this repeat is 10.2 bp. If theDNA were relaxed with an intrinsic helical repeatof 10.5 bp this implies that FIS will induce the for-mation of a left-handed superhelical loop becausethe helical spacing of the binding sites is less thanthe helical repeat of DNA. However the relativerotation between binding sites will vary with thedifference between this local helical repeat and theintrinsic helical repeat of the DNA. This lattervalue itself depends on the unconstrained superhe-lical density. In other words, given a requirementfor a precise binding geometry variations in super-helical density may alter the af®nity of DNA forproteins by altering the geometry of DNA. Forexample, negative superhelicity has been shown tolower the initial binding af®nity for RNA polymer-ase at the tet promoter (Bertrand-Burgraff et al.,1984).

The role of DNA superhelix densityand promoter design

We have argued that the function of FIS in atranscriptional context is to overcome impedimentsto initiation that reduce the overall rate of poly-merase turnover at stable RNA promoters. Thisleaves unanswered the question of the nature ofthe normal physiological block. Recent evidencesuggests that a major role of FIS in vivo is to act asa topological ``homeostat'' (Schneider et al., 1997).FIS production is maximal at the transition fromstationary to exponential phase (Ball et al., 1992;Ninnemann et al., 1992). At this time after a pro-longed period of stationary growth DNA plasmidsexist as two major discrete topological populations,one of intermediate and the other of high negativesuperhelical density (Schneider et al., 1997). In ®s�

cells the former population is maintained untilmid-exponential phase whereas in ®sÿ cells it israpidly converted to more highly supercoiledforms. There are thus two important variableswhich affect ®s-dependent promoter activity: thesuperhelical density of the template DNA and theoccupation of the FIS binding sites in the UAS.In vitro the activity of the tyrT promoter is stronglydependent on negative superhelicity (Lamond,1985) as it is in vivo (Free & Dorman, 1994). How-ever, in vivo tyrT transcription becomes less depen-dent on the UAS at high negative superhelicaldensities (A. Deufel, H. Auner, L. Lazarus, A.T. &G.M., unpublished observations). The relationshipof the transcription activation function of FIS tosuperhelical density is further strengthened by theobservation that changes in DNA superhelicity

induced by DNA gyrase inhibitor novobiocinin vivo affect rrnB P1 expression only when FIS isnot present (M. Aviv & G. Glaser, personal com-munication). Similarly in vitro FIS preferentiallystimulates nrd transcription at lower unconstrainedsuperhelical densities (Sun & Fuchs, 1994). Takentogether these observations suggest that the lowerlevels of superhelical density in ®s� cells are insuf-®cient to support ef®cient stable RNA synthesisand thus that one function of FIS bound to UASregions is to compensate locally for a generalreduction in negative superhelicity. This could beaccomplished indirectly, by, for example, con-straining the UAS in a microloop of higher super-helical density than the available unconstrainedvalue. This would require the participation of allthree FIS sites.

In summary we envisage that the microloopsformed by the rrnB P1 and tyrT UAS regionsenable high rates of initiation under conditions ofintermediate superhelical density but at highsuperhelical densities they become dispensable. Inthis scenario FIS acts both to lower the overallsuperhelical density in vivo (Schneider et al., 1997)and to stabilise the compensating UAS micro-loops. This dual action results in the activationof a particular subset of supercoiling-dependentpromoters.

The activation by FIS of different stepsin the initiation process

A further question is which steps in the initiationprocess are facilitated by FIS in vivo. The absolutelevel of transcription from the plasmid-borne wild-type tyrT promoter is essentially ®s-independentin vivo (Lazarus & Travers, 1993). However downmutations in the ÿ10 hexamer of both tyrT andtufB promoters confer ®s-dependence (Lazarus andTravers, 1993) as also do ``up'' mutations in theÿ35 hexamer, the ÿ35 to ÿ10 spacer and the discri-minator (A. Deufel, H. Auner, L. Lazarus, A.T. &G.M., unpublished observations). Nevertheless ®s-independent promoter activity can be restored at a2.5 to threefold higher level than the wild-typepromoter by a combination of up mutations in thespacer and discriminator. Thus for the tyrT promo-ter there is the apparent paradoxical situation inwhich FIS stimulates transcription from mutantpromoters with either enhanced or diminishedpotential activity but does not, under normal lab-oratory conditions, signi®cantly affect transcriptionfrom the wild-type promoter. This contrasts withthe usual regulatory situation in which ``up''promoter mutations normally reduce the extent ofregulation, e.g. the lac ps and UV5 mutations in theÿ10 hexamer (Gilbert, 1976). However, thesein vivo observations are consistent with the abilityof FIS to facilitate sequential steps in the initiationprocess. Thus a ÿ10 down mutation is expected toimpair the binding of polymerase and thus hinderthe nucleation of untwisting, an effect which could


be overcome by an increased residence time forpolymerase in the initial complex. This can beachieved either by FIS-dependent stabilization ofthe microloop or by a mutation of the ÿ35 regionto the consensus sequence (Lazarus & Travers,1993). Conversely the same ÿ35 up mutation inthe wild-type context reduces promoter activityin vitro, particularly at higher superhelical densities(A. Deufel, H. Auner, L. Lazarus, A.T. & G.M.,unpublished observations). In a study of syntheticpromoters Ellinger et al. (1994a) showed that highhomology to the consensus sequence resulted inimpaired promoter clearance. If this is also true forthe mutant tyrT promoter it implies ®rst, that thefacilitation of polymerase escape by FIS observedin vitro (Muskhelishvili et al., 1997) may also beoperating in vivo and second, that increasing super-helicity may have a negative effect on promoterclearance, possibly analogous to the increase inpolymerase pausing during RNA chain elongationon supercoiled templates (Krohn et al., 1992). Simi-larly an increase in the spacer from 16 to 17 bpdecreases promoter activity at high superhelicaldensities even though in other contexts the samechange increases activity (Stefano & Gralla, 1982).The inference is that optimal promoter designdepends strongly on superhelical density. There isalso substantial evidence that a primary role ofnegative superhelicity is to promote the DNAuntwisting associated with promoter opening atstable RNA and other promoters. The preciseresponse depends on individual promoter architec-ture but in general an enhanced response toincreasing superhelical density is apparent inpromoters with suboptimal ÿ35 or ÿ10 hexamers,suboptimal spacers and G/C-rich discriminatorregions (Borowiec & Gralla, 1987; Giladi et al.,1992; Jordi et al., 1995). We note that if increasingnegative superhelicity favours promoter openingbut disfavours escape this could provide a ration-ale for the general role of FIS as a topologicalhomeostat. Thus there are at least two ways to alle-viate the effects of suboptimal unconstrainedsuperhelical densities at stable RNA promoters byFIS: by increasing the initial residence time of poly-merase to compensate for the suboptimal ÿ35region and by directly facilitating promoter open-ing and polymerase escape by torsional trans-mission.

We surmise that initiation at the wild-type tyrTpromoter is ®nely tuned so that under optimumconditions (i.e. at optimal unconstrained superheli-cal densities) the different steps in the initiationprocess are kinetically coordinated, i.e. no one stepis strongly rate-limiting. The role of FIS in such asituation would be to act as a facultative activatorovercoming any kinetic bottlenecks caused by vari-ations in superhelicity or by polymerase limitation.Such a role would ensure the maximal rates ofinitiation essential for stable RNA production.A direct experimental approach to this problemwould be the analysis of the activating potential of

FIS using the promoter constructs on plasmidsdiffering in superhelical density.

It is important to note that this discussion isbased on observations from a small sample ofpromoters. Given the apparent variability in thequality, number and position of FIS binding sitesupstream of the core promoter of stable RNAgenes it seems probable that individually such pro-moters may respond differently to FIS, particularlywith respect to the relative effects on differentsteps of the initiation process.

Other sigma70-dependent promoters

We have proposed that the UAS of stable RNApromoters functions by forming a microloop whichacts as a torsional store for driving promoter open-ing and polymerase escape. In this section we shalladdress the question of whether this mode of acti-vation by torsional transmission is peculiar tostable RNA promoters or is a particular adaptationof a mechanism that is utilised by most, or perhapsall, s70-dependent promoters.

We make the assumption that the mechanisticaspects of core polymerase function are essentiallythe same at all types of promoter. This implies thatthe formation of the intermediate complex at, forexample, the T7 A3 promoter requires a relativerotation of two contiguous regions of DNA (seeFigure 2B). In the absence of an extensive upstreamregion the torsion generated could be accommo-dated in two regions, the ÿ35 to ÿ10 spacer andthe ÿ10 region itself where untwisting is facilitatedby the presence of the ¯exible TA base-steps. Thisaccommodation can be enhanced in vitro, particu-larly in the ÿ10 region, by the introduction ofsingle-strand gaps which increase the ¯exibility ofDNA (Werel et al., 1991; NeÁgre et al., 1997). Anexample of this ¯exibility is a DNase I hypersensi-tive site within the spacer region, present as a pro-minent structural feature in the intermediate andopen complexes of many promoters with 17 and18 bp spacers, but normally absent from stableRNA promoters with a 16 bp spacer. In the opencomplex at the lac promoter DNA in the vicinity ofthis DNase I sensitive site is sensitive to cleavageby singlet oxygen (Buckle et al., 1992). This reagenttargets deformed untwisted regions of DNA. Theimplication is that the DNA in the spacer regionsof these promoters, but not that in the 16 bp spacerof some stable RNA promoters, is untwisted, asoriginally suggested by Stefano & Gralla (1982).However, promoter function is not correlated withthe inferred extent of spacer untwisting as opencomplex formation is facilitated when both 16 bpand 18 bp spacers are changed to 17 bp (Borowiec& Gralla, 1987; Ayers et al., 1989; Jordi et al., 1995),consistent with the notion that there is an optimalalignment of the ÿ35 and ÿ10 hexamers in theopen complex. By contrast the optimal spacerlengths for initial complex formation are 16 and17 bp (Ayers et al., 1989), again suggesting a


change in the rotational alignment on transition ofthe initial complex to the open complex.

Does the deformation in the spacer contribute topromoter opening and/or polymerase escape?A comparison of 17 bp spacer promoters with andwithout a gap in the spacer showed that gappingreduced the rate of initiation complex formation(kf) by at least fourfold (Ayers et al., 1989). Thisproperty is consistent with a direct facilitation ofpromoter opening by the torsion in the spacer.However in the same experiments gapping did notalter kf for promoters with either 16 bp or 18 bpspacers. These promoters contained an optimalÿ35 hexamer but for the proU promoter, with asub-optimal ÿ35 hexamer, a nick in one strand ofthe 16 bp spacer promotes open complex for-mation (Jordi et al., 1995). One possible explanationfor this discrepancy is that the optimal ÿ35 hexam-er restricts the dynamic function of polymerase bya strong anchoring effect. This can allow simul-taneous contacts to the ÿ35 and ÿ10 hexamerswith the optimal 17 bp spacer. A gap in the 17 bpspacer is expected to increase the rotational ¯exi-bility of DNA between the hexamers and wouldthus have a negative effect. By contrast, gappingwould have a small effect with 16 bp and18 bp spacers, where the simultaneous contactswith ÿ35 and ÿ10 hexamers are primarilyrestricted by suboptimal spacing, rather than byrotational ¯exibility. However, if the ÿ35 regionis suboptimal the anchoring effect would beweak, allowing for better adjustment of poly-merase to the gapped ÿ16 spacer, presumablybecause the enhanced ¯exibility favours for-mation of an optimal rotational alignment ofthe ÿ35 and ÿ10 hexamers. Thus the evidencefor an active role for torsion in the spacer isnot yet conclusive while the effect of spacerlength and torsion on polymerase escape is sofar unexplored. To test this hypothesis an anal-ysis of several combinations of intact comparedwith gapped spacers in the context of promo-ters with optimal versus suboptimal ÿ35 regionis necessary.

Mechanisms of activation bytranscription factors

The recruitment of RNA polymerase by a DNA-bound activator or activator complex is a straight-forward and often cooperative interaction betweenthe two components in which the factor providesan extended binding site for the polymerase eitherby protein-protein contacts or by bringingupstream DNA into close proximity with a second-ary DNA binding site on RNA polymerase. Bycontrast the mechanisms by which transcriptionfactors can facilitate subsequent steps in theinitiation process are relatively poorly understood.However, an inevitable consequence of the for-mation of a ternary complex between polymer-ase, activator and promoter DNA is the

formation of additional DNA microdomains,both between the activator and the polymerasebinding site and in some cases between the acti-vator and the backside of the polymerase. Forsuch microdomains to function as topologicallyindependent entities either a microloop delimitedby polymerase must be formed or the proteincoupling between the activator and the polymer-ase must be suf®ciently rigid to prevent signi®-cant rotation of one protein relative to the other(DeÂthiollaz et al., 1996).

The importance of the functional integrity ofactivator-dependent microdomains is illustrated bythe spacer between the CRP binding site and theÿ35 region of the lac promoter. Within this regionthere is a CRP-dependent singlet-oxygen sensitivesite at position ÿ46 (Buckle et al., 1992). In otheractivator-promoter combinations (IHF at the l pLpromoter (Giladi et al., 1992) and FIS at the tyrTpromoter (Muskhelishvili et al., 1995)) a similarDNA distortion in the activator-polymerase spaceris apparent from hypersensitivity to DNase I clea-vage. When the sugar-phosphate backbone of theCRP-polymerase DNA spacer in the lac promoteris interrupted by a single break activator functionis lost even though polymerase-CRP protein-pro-tein contacts are maintained (Ryu et al., 1994). Simi-larly at the malT promoter the DNA geometry ofthe upstream region is crucial for the facilitation ofpolymerase escape by CRP (DeÂthiollaz et al., 1996).By contrast to the lac promoter, binding of CRP atposition ÿ41.5 at the gal P1 promoter enhancesboth polymerase recruitment and promoter open-ing (Herbert et al., 1986). Here CRP could compen-sate for lack of a ÿ35 region by delimiting themicrodomains normally established by RNApolymerase.

In all of the above examples the rotational orien-tation of the activator with respect to polymerasemust be conserved for activation to occur. Yetthere are other examples which apparently do notobey this rule. The activation of the ilvPG promoterby IHF is less dependent on rotational orientationof the activator protein relative to the polymerase(Parekh & Hat®eld, 1996), a shift of 6 bp in theupstream binding site of IHF resulting only in areduction of activation from fourfold to two-fold. IHF can also be functionally replaced bythe eukaryotic DNA-bending protein LEF-1,suggesting that direct activator-polymerase con-tacts are not required in this case. The authors pro-pose that IHF structurally alters the DNA helix ata distance in a way that facilitates open complexformation at the downstream ilvPG promoter siteand present evidence that IHF induces such analteration in the ÿ10 region in the absence of poly-merase. The latter observation implies long rangeinteractions and at least transient formation of atopologically closed domain. However, theobserved small reduction in activation by a shift of6 bp does not necessarily exclude microloop for-mation. We note that if IHF were to induce theformation of a polymerase-bounded microloop


capable of rotation relative to another polymer-ase-bounded microdomain the lack of face-of-the-helix dependence of IHF binding could simplyre¯ect different preferred conformations of themicroloop.

One consequence of the formation of microdo-mains is that an activator such as FIS which nor-mally forms a microloop will, by virtue of amicrodomain between the polymerase and theimmediately proximal bound activator, retain atleast a partial activating function even if the loopwere not completely formed. In such a situationactivation by torsional transmission does notrequire complete occupancy of multiple bindingsites (Muskhelishvili et al., 1997), especially in vitrowhere it is possible to alter the energy requirementfor promoter opening and polymerase escape byvarying such parameters as salt concentration andtemperature. Nevertheless, an important indicatorof the role of DNA geometry in such promoters isthe in vivo occupancy of multiple sites. In the caseof the rrnB P1 promoter the available evidencesuggests that occupancy of the three FIS bindingsites is maintained in vivo even though a single FISdimer bound at the proximal FIS binding site issuf®cient for polymerase recruitment in vitro(Bokal et al., 1995).

Transcriptional activation ofholoenzymes containing alternativesigma factors

So far we have considered s70-dependentinitiation. It is reasonable to ask to what extentinitiation directed by other sigma factors is compa-tible with the concept of torsional transmission.

One particularly instructive example is providedby the late genes of E. coli bacteriophage T4. Theseare transcribed from a very simple promoter, con-sisting of a single recognition sequence TATAAA-TA, centred at ÿ10 relative to the transcriptionstart. This sequence is recognised by a phage-encoded sigma factor, gp55, in association with thehost core polymerase but with these proteins alonethe rate of promoter opening is slow (Sanders et al.,1997). However the rate of promoter opening isincreased approximately 100-fold in the presenceof two other proteins gp33 and gp45. Gp45 is theT4-encoded homologue of PCNA in eukaryotesand of the b subunit of E. coli DNA polymerase IIIand acts as a sliding clamp that tracks along theDNA in association with the replication complex.By contrast gp33 is a prototypical transcriptionalcoactivator that is required for activation in thepresence of gp45 but represses basal transcriptionin its absence (Herendeen et al., 1990). The proper-ties of these proteins account for the dependence oflate T4 transcription in vivo on concurrent DNAreplication (Riva et al., 1970). A further crucial fea-ture is the requirement for an unobstructed path-way on the DNA between the loading site for gp45and the promoter (Geiduschek et al., 1997). This

implies the existence of a con®ned DNA domainbetween the clamp and the polymerase. Althoughit is plausible that negative superhelicity could begenerated in the loop by the progression of thesliding clamp, there is no evidence for the existenceof a looped structure. Indeed cross-linking dataindicate that gp45 is located in the transcriptioncomplex between 31 and 41 bp upstream of thestartpoint (Tinker et al., 1994). Both gp45 and gp33contact RNA polymerase and act synergistically toincrease the rate of promoter opening and alsopossibly to facilitate polymerase recruitment(Tinker-Kulberg et al., 1996; Sanders et al., 1997).Gp45 could thus act in an analogous manner toCRP at the gal P1 promoter (Herbert et al., 1986)and compensate for the lack of a de®ned secondgp55 binding site upstream of the ÿ10 recognitionsequence i.e. gp45 would anchor the upstream endof the polymerase. Alternatively gp45 could itselfgenerate torsion by rotating on the DNA.

Another ubiquitous class of promoters are thosedependent on s54, which is structurally unrelatedto s70. s54 promoters contain two recognitionelements but these, at ÿ10 and ÿ24, respectively,are separated by one helical turn less than the ÿ10and ÿ35 hexamers in s70 promoters. In the absenceof activator the s54-containing holoenzyme formsclosed complexes at its cognate promoters. Thetransition to the open complex is completelydependent on interaction with activator and forone class of activators including NtrC, XylR andDctD is coupled to ATP hydrolysis and phos-phorylation (Keener & Kustu, 1988; Weiss et al.,1992). A second class of activators, including NifA,although otherwise homologous to NtrC lacks theN-terminal domain containing the phosphorylationsite (Drummond et al., 1986; Lee et al., 1993). Theactivator protein is normally bound to a site �100to 200 bp upstream of the transcription startpointand in the activated ternary complex in the pre-sence of ATP constrains a DNA microloop (Su et al.,1990). ATP hydrolysis induces formation of theopen complex and the breaking of the microloop(Rippe et al., 1997). In an analogous manner to s70

promoters open complex formation is accompaniedby an extension of the polymerase footprint from�1 to �20 (Popham et al., 1989). However, in vitrothe transition to the open complex can be inducedby high concentrations of mutant activator whichcan no longer bind to DNA (Berger et al., 1994).This observation implies that a major role of micro-loop formation by curved DNA (Carmona &Magasanik, 1996) or by IHF (Hoover et al., 1990;Claverie-Martin & Magasanik, 1991) is to bringactivator and holoenzyme into close and appropri-ate spatial proximity (Wedel et al., 1990) but doesnot exclude an additional role for torsionaltransmission. One situation in which torsionaltransmission via DNA may play little, if any,role in promoter opening at these promoters iswhen activation occurs from sites up to 1-2 kbfrom the core promoter (Reitzer & Magasanik,1985), a distance which would allow facile


redistribution between untwisting and writhe.These observations imply strongly that theactivator directly induces a conformational tran-sition in RNA polymerase through protein-pro-tein contacts.

The role of sigma factors

We have argued that the detailed mechanism bywhich torsion is generated and utilized differs ats70 and s54-dependent promoters. For the mostactive s70-dependent promoters we suggest thatthe torsion generated by a conformational changein the holoenzyme is stored in a DNA microloopand can be subsequently utilized to drive promoteropening. By contrast at s54-dependent promotersthe equivalent conformational transition in poly-merase is driven by activator-dependent NTPhydrolysis, presumably involving a conformationalchange in the activator. However in both situationsthe prime requirement is that there be a rigidcontact between the activating element, be it DNAor protein, and the polymerase. The availableevidence also indicates that the sigma subunit is anintegral part of the mechanism of this ``general''torsional transmission. Thus the s54 activator DctDcrosslinks to amino acids that are surface exposedand connect the two DNA binding domains of thesigma factor (Lee & Hoover, 1995). Similarly s70

can directly interact with FIS (Muskhelishvili et al.,1995), in addition to contacts with the corepolymerase (G.M. & M. Buckle, unpublished obser-vations; Bokal et al., 1997) and the sigma factor forlate T4 transcription, gp55, interacts directly withthe activator gp45. Further for both FIS and CRPthere is no evidence that they undergo confor-mational changes during the activation process,although such transitions cannot be rigorouslyexcluded. We suggest that the transition from theinitial to the intermediate complex is determinedby the interactions of the sigma subunit with thecore polymerase and that the detailed mechanismat a given promoter is dependent on the type ofsigma factor and the promoter organisation. Thisimplies that at FIS-dependent promoters, forexample, the torsion generated by a s70-dependentconformational change in polymerase is trans-mitted to the microloop via FIS. Conversely,because the system is topologically closed any tor-sion stored or trapped in the loop by FIS will becoupled to the conformational changes in RNApolymerase as well as to subsequent initiationevents.

Mechanistic parallels betweentranscription initiation andDNA inversion

In addition to facilitating transcription initiationFIS is also an essential component of the so-calledsynaptic complex which supports the site-speci®cDNA inversion reactions catalysed by closely

related Gin, Hin, Cin and Pin invertases (for areview see van de Putte & Goosen, 1992). Are theroles of FIS in these two disparate nucleoproteincomplexes mechanistically related? In commonwith stable RNA transcription, the ef®ciency ofDNA inversion strongly depends on the superheli-cal density of DNA (Lim & Simon, 1992; Benjaminet al., 1996). It has been proposed that negativesupercoiling both favours the speci®c alignment ofthe recombination sites before DNA strand clea-vage and also imposes directionality on therotation of cleaved strands (Kanaar et al., 1990;Moskowitz et al., 1991; Klippel et al., 1993) whichin turn is coupled to the dissipation of the freeenergy of negative supercoiling (Stark et al., 1989).In these systems binding of FIS to multiple sites inthe cis-acting recombinational enhancer element(Kahmann et al., 1985; Johnson & Simon, 1985;Huber et al., 1985) stabilises a speci®c nucleopro-tein assembly in which two dimers of invertasebound to inversely (head to head) oriented recom-bination sites are thought to tetramerise in a pro-cess termed synapsis. The assembly of the synapticcomplex is a prerequisite for double-stranded clea-vage of crossover sites. This scission is followed byrotation of the duplexes by 180� and their religa-tion in recombinant con®guration (Kanaar et al.,1990). It is thought that the synaptic complexforms at a branch point in DNA by simultaneousintertwining of the FIS-bound enhancer with tworecombination sites bound by the invertase tetra-mer (Kanaar et al., 1989). The FIS-enhancercomplex plays the role of a ``topological ®lter'' pro-viding for the formation of a topologically uniquesynapse (Crisona et al., 1994). Several lines ofevidence suggest that in addition to this architec-tural role, FIS also facilitates unwinding of DNAwithin the crossover sites and causes a confor-mational rearrangement of invertase moleculesleading to a concerted double-strand cleavage ofrecombination sites (Klippel et al., 1993; Haykinsonet al., 1996; Deufel et al., 1997; G.M., unpublishedobservations).

Is there any similarity in the structural organis-ation of the FIS-UAS and FIS-enhancer complexes?Not only does FIS bend the enhancer (Perkins-Balding et al., 1997) but the extent of the enhancersequences (60 to 90 bp) is comparable to that of theUAS regions and, by analogy to the rotational con-straints governing the function of FIS-UAS com-plexes, the FIS-enhancer complex must have aspeci®c spatial orientation with respect to the prox-imal recombination site (Rudt, 1990; Haykinson &Johnson, 1993). Furthermore, the high-af®nity FISbinding sites in the enhancer are conserved innumber and helical arrangement, as insertionsdisrupting the helical phasing between these sitesimpair the stimulatory effect of FIS on DNA inver-sion (Johnson et al., 1987; HuÈ bner et al., 1989; Rudt,1990). This observation parallels the impairment ofthe tyrT UAS function by a 5 bp insertion whichdisrupts the helical phasing of the three FIS bind-ing sites. The bending of the enhancer induced by


FIS, probably in conjunction with intrinsic curva-ture of DNA (Perkins-Balding et al., 1997), resultsin a speci®c ``active'' conformation of the enhancer.However, DNA bending by binding of FIS to highaf®nity sites does not suf®ce for the induction ofthe active enhancer conformation. The interveningregion between the FIS binding sites also plays arole in this process, since even single base substi-tutions in this region suf®ce to inactivate theenhancer (HuÈ bner & Arber, 1989). It is possiblethat in the Hin and Cin enhancers the energyrequired for the induction of an active enhancerconformation is supplied by a non-speci®c bindingof a third FIS molecule between the two FIS dimersbound at high af®nity sites (Haffter & Bickle, 1987;HuÈ bner et al., 1989). Likewise, the Gin enhancercontains three FIS binding sites, site III being oflower af®nity than sites I or II (Rudt, 1990). Alter-natively the activation of the enhancer occurs onlyafter physical coupling of the FIS-enhancer com-plex with invertase.

The separations between the high af®nity FISsites I and II in the Hin enhancer and between sitesI, II and III in the Gin enhancer are close to mul-tiples of 12 bp suggesting that the architecture ofFIS-induced loops is different from that of the tyrTUAS. In relaxed enhancer DNA the FIS dimersshould bind to opposite faces of the DNA helix

Figure 5. Proposed stimulation of DNA inversion bytorsional transmission. A, Binding and bending of DNAby FIS bound to the Gin enhancer initially forms a topo-logically neutral zigzag. B, Rotation of the DNA micro-domain between bound FIS dimers results in theformation of a right-handed microloop. C, Coupling amicrodomain rotation to structural rearrangement of theGin invertase tetramer.

(Johnson et al., 1987). The resultant bending ofDNA then induces a topologically neutral kink(Figure 5A). Another possibility is that FIS inducesa coherent bend in enhancer DNA, but the handed-ness of the respective microloops in the recombina-tional enhancers and in the transcriptional UASregions differ: the helical repeat of the FIS bindingsites in the UAS is 10.2 to 10.3 bp and is lowerthan the repeat of relaxed DNA (�10.5 bp)whereas that of the FIS binding sites in the enhan-cer is higher. If it is assumed that in both cases theloop approximates to wrapping on a cylindricalsurface this implies that the FIS binding sites in theUAS de®ne a left-handed loop and those in theenhancer de®ne a right-handed loop (Figure 5B).We have argued that torsional transmission to anindependent microdomain requires a confor-mational transition in the microloop. We suggestthat the con®guration imposed on occupation ofhigh af®nity FIS binding sites in the enhancer dif-fers from that preferred in the presence of invertaseor additional FIS dimers bound to secondary sites.Protein-protein interactions between the FIS-enhan-cer complex and invertase might directly contrib-ute to the rotation of the enhancer microloop bybringing the two bends induced at high af®nitysites into approximate register (Figure 5C and D).In a coherently bent right-handed enhancer micro-loop the accommodated torsion could be stored intopologically isolated microdomains delimited byFIS-DNA contacts and by protein couplingbetween FIS and the invertase (Deufel et al., 1997).Physical association of these proteins bound totheir cognate sites at a branch point in supercoiledDNA would allow transmission of the torsionaccommodated in the enhancer microloop viaconformational rearrangements induced in inver-tase tetramer to crossover sites facilitating theiruntwisting and supporting high rates of recombi-nation. We note that the microloop structured byFIS at the enhancer is essentially independent ofthe topology of ¯anking DNA, since FIS can stimu-late inversion even if the enhancer is located on aseparate nicked DNA ring in a catenane (Benjaminet al., 1996). This indicates that a primary require-ment for negative superhelicity is to facilitate aninvertase-mediated function. We suggest, byanalogy to transcription initiation, that thisfunction is the untwisting of DNA.

The generation of torsion

The mechanism of generation of torsion can bebest explained on the example of the microloopsconstrained by FIS. For both FIS-dependent DNAinversion and promoter opening we postulate thatan essential requirement for torsional transmissionis a repartitioning of twist and writhe within themicroloop stabilised by FIS. Such a change in thegeometry of the microloop implies a conformation-al ¯exibility which can be achieved if either thecontacts between FIS and DNA or between the FIS


dimers can be varied. FIS-DNA contacts could bevaried for example, by changing the number ofoccupied sites or by making or breaking weak con-tacts between FIS and ¯anking DNA (Pan et al.,1996). Another, not mutually exclusive, possibilityis that adjacent DNA-bound FIS dimers couldassume different contact con®gurations asobserved in crystals of different forms of the pro-tein (Theis, 1996). The accommodation of changingtopological DNA con®gurations by FIS wouldallow any intrinsic rotation of polymerase or inver-tase to be transmitted via rigid coupling to themicroloop and vice versa. Such a mechanism wouldalso enable FIS to contribute to the generation oftorsion necessary for the nucleation of untwistingand so compensate locally for low levels of nega-tive supercoiling at stable RNA promoters. Theimplication is that FIS itself provides the additionalenergy for this nucleation. In this respect activationby FIS is analogous to that by activators of s54

promoters.

Conclusion

This review represents an attempt to unify thegreat diversity of mechanisms of prokaryotic tran-scriptional activation by introduction of the con-cept of torsional transmission. This concept putsforward the view that in most, if not all systemsdescribed so far, physical coupling between pro-teins and between proteins and DNA in enzymati-cally active large complexes results in theformation of topologically closed domains whichare capable of storing and utilising torsion. Thestored torsion is used by effecting enzymes to over-ride different thermodynamic and kinetic barriersto the reaction. The transmission of the torsionalenergy is provided by tight coupling between thecomponents of the topological domain in conjunc-tion with the conformational ¯exibility of protein-protein and/or protein-DNA contacts. The DNAwithin a closed topological domain is often furthersubdivided into functionally distinct microdomainsdelimited by multiple protein-DNA contacts withinhigher-order nucleoprotein complexes. The tor-sional strain stored by such nucleoprotein com-plexes is partitioned between these microdomainsand can be released in discrete steps, thus formallyresembling the stepwise release of energy in cata-bolic enzymatic reactions.

Acknowledgements

We thank all our colleagues who have contributed tothis work during the past years. We also thank Dr G.Glaser and Dr Malcolm Buckle for communicatingunpublished results. This work was in part supported bythe Deutsche Forschungsgemeinschaft through SFB 190.

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Edited by M. Gottesman

(Received 3 September 1997; received in revised form 10 March 1998; accepted 20 March 1998)

DNA microloops and microdomains: a general mechanism for transcription activation by torsional...

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