The EMBO Journal vol.9 no.7 pp.2215 - 2220, 1990

Topography of intermediates in transcription initiation ofE.coli

Peter Schickor, Willi Metzger, WiadyslawWerel1, Hermann Lederer and HermannHeumann

Max Planck Institute of Biochemistry, D-8033 Martinsried, FRG and'University of Gdansk, Department of Molecular Biology, 80-822Gdansk, ul. KLadki 24, Poland

Communicated by W.Zillig

Three characteristic footprinting patterns resulted fromprobing the Escherichia coli RNA polymerase T7 Alpromoter complex by hydroxyl radicals in thetemperature range between 4°C and 37°C. These wereattributed to the closed complex, the intermediatecomplex and the open complex. In the closed complex,the RNA polymerase protects the DNA only at one sideover five helical turns. In the intermediate complex, therange of the protected area is extended further down-stream by two helical turns. This region of the DNA helixis fully protected, indicating that the RNA polymerasewraps around the DNA between base positions -13 and+20. In the open complex, a stretch between basepositions -7 and +2, which was fully protected in theintermediate complex, becomes accessible towardshydroxyl radicals but only in the codogenic strand,indicating that the DNA strands are unwound. Our datasuggest that only the DNA downstream of the promoteris involved in this unwinding process.Key words: Escherichia coli/footprinting/transcription

IntroductionFormation of a transcription competent complex by DNAdependent RNA polymerase (R) and a promoter sequence(P) is preceded by at least two intermediates. The reactionleading to the initiation complex (RP0) can be described atleast for strong promoters by the following scheme:

R + P = RPc RPi = RPO (1)

where RPc is the closed complex, RPi an intermediate andRPO the open complex, in which the two DNA strands areseparated (Walter et al., 1967; Chamberlin, 1976). Thisreaction scheme was proposed on the basis of results obtainedfrom kinetic studies (Hawley et al., 1982; Rosenberg et al.,1982; Buc and McClure, 1985; Roe et al., 1985; Spasskyet al., 1985). At 37°C the complexes RPc and RPi are shortliving intermediates not accessible to biochemical analysis.There is good evidence from kinetic studies that thesetransient intermediates can be accumulated within a giventemperature interval; the closed complex RPc below 8°C,the intermediate complex RPi between 8°C and 21°C and

the open complex RPO above 21°C (Kirkegaard et al.,1983; Straney and Crothers, 1985). These three complexesdiffer in their sensitivity to heparin and in their ability toform an open complex. RP, is heparin sensitive whereasRPi and RPO are heparin insensitive (Schickor, 1990). TheDNA within the promoter region of RPc and RPi is in theclosed-form. Only the DNA within the promoter region ofthe RPO is separated (Kirkegaard et al., 1983) between basepositions -10 and +2 (Siebenlist, 1979). Strand separationis correlated with an unwinding of the DNA (Wang et al.,1977; Gamper and Hearst, 1982a and b; Amouyal and Buc,1987). This unwinding is temperature dependent, startingabove 21°C as shown by Kirkegaard et al. (1983).The complexes RPC, RPi and RPO have been intensively

investigated by footprinting analysis (Simpson, 1979;Siebenlist and Gilbert, 1980; Hofer et al., 1985; Spasskyet al., 1985; Buckle and Buc, 1989). The probes used inthese previous studies were either only base specific orenzymatic, therefore, these results cannot be usedconclusively to determine the structure of the RNApolymerase-promoter complex. Hydroxyl radicals showonly slight base specificity (Tullius and Dombroski, 1986).The DNA 'footprint' obtained with this probe is a directmeasure of the accessibility of a DNA in a protein-DNAcomplex. We have used hydroxyl radicals to probe the phageT7 promoter Al complexed with Escherichia coli RNApolymerase. The aim of these studies is to show if and howthe regions of the DNA protected by RNA polymerasechange at different temperatures. This information combinedwith structural information from previously publishedneutron scattering studies (Heumann et al., 1988), was usedto obtain a more detailed model of the RNA polymerase-promoter complex.

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Fig. 1. Acrylamide gel electrophoresis of the complex of RNApolymerase and a DNA fragment of 130 bp carrying the Ecolipromoter Al of the phage T7, under non-denaturing conditions. Thetemperature was a linear gradient ranging from 20C to 40'C.

2215© Oxford University Press

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Fig. 2. Electrophoretic analysis of the hydroxyl radical footprints ofRNA polymerase-promoter complexes at the temperatures T = 4°C(lane 2), T = 8°C (lane 3), T = 20°C (lane 4), T = 30°C (lane 5),T = 37°C (lane 6). Lane 1 shows the DNA without RNApolymerase. Panel A displays the footprints of the non-codogenicstrand and panel B of the codogenic strand.

ResultsThe E. coli RNA polymerase complexed to the 'strong' Alpromoter of the phage T7 is stable at low ionic strength ata temperature range from 4°C to 37°C as shown by the bandshift assay in Figure 1. Complexes formed at the varioustemperatures between 4°C and 37°C (see Figure 2) weresubjected to hydroxyl radical treatment as described inMaterials and methods. These complexes were separatedfrom free DNA by electrophoresis under non-denaturingconditions (Straney and Crothers, 1985; Heumann et al.,1986), eluted, and analysed on a sequencing gel. Theresulting protection patterns of the 3'- and 5'-end-labelledstrands are shown in Figure 2. Figure 3 shows the absorptionscans of the footprints at the codogenic strands, whereasFigure 4 is a schematic representation of the results.Three distinct footprinting patterns were observed in the

temperature range from 4°C to 37°C which could beassigned to the closed complex (<8 C), the intermediatecomplex (8°C-21 °C) and the open complex (> 21 C). The

pattern of the closed complex reaches from the base positions-4 to -53. Within this region the DNA is partially protectedand shows a periodic change of the pattern. This indicatesthat the RNA polymerase faces towards one side of theDNA. This interpretation is supported by the finding thatboth DNA strands show the same pattern with an offset ofthree bases as expected for a protein bound on one side ofa helical structure (Tullius and Dombroski, 1986). Theperiodicity of the pattern is on average 10.6 bases perhelical turn, close to the value expected for a DNA in B-conformation. This implies that upon binding of RNApolymerase to the promoter DNA, there is no significantdistortion of the B-DNA conformation in this region.Above 12'C, in the intermediate complex, the protected

area is extended further downstream by about two helicalturns reaching from base position -53 to position +21.The pattern may be subdivided into two parts, the regionupstream of base position -14, called the 'recognitiondomain' (Metzger et al., 1989), which remains unchangedcompared with the closed complex, and the region down-stream of base position -13, which shows a distinctdifference. This 'melting domain' (Metzger et al., 1989)reaches downstream to base position + 18 in the codogenicstrand and to +21 in the non-codogenic strand, and is fullyprotected (with the exception of the bases -10 and -11 inthe non-codogenic strand), indicating that this part of theDNA is in close contact with the protein.The pattern characteristic for the open complex appears

2216

Footprinting of transcription complex

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complexes (RP0).

when the temperature is increased above 20°C. It isessentially the same as found for the intermediate complex,except that the sequence between base positions -7 and +2becomes sensitive towards hydroxyl radicals, and only inthe codogenic strand. The non-codogenic strand remainsfully protected in this domain (Figure 4), suggesting that bothDNA strands are separated. The size of the region that isfully accessible to hydroxyl radicals consists of about ninebases centred at base position -4. To a lesser extent, theregion further downstream of the transcription bubblebetween base positions +2 and + 12 is also accessible tohydroxyl radicals as the comparison of the scans for the 20°Ccomplex and the 37°C complex suggests (Figure 3). If thehypothesis that accessibility of the codogenic strand withinthe melting domain can be assigned to strand separation iscorrect, this process should be essential for the formationof an active transcription complex. Therefore, we determinedthe relative amount of transcription complexes by measuringthe yield of RNA synthesizing complexes (ternarycomplexes). Figure 5 shows the yield of ternary complexesin the temperature range between 20°C and 37°C. Bothprocesses, the accessibility of the DNA in the codogenicstrand and the formation of a transcription competentcomplex, have the same temperature dependence.

DiscussionWe have chosen hydroxyl radicals in order to obtainstructural information about the complex ofDNA dependentRNA polymerase and promoter DNA. The resolutionachieved by 'footprinting' with hydroxyl radicals isdetermined by the distance of neighbouring bases, which is0.33 nm, if the probed DNA is in the B-conformation. Thisinformation is, however, restricted to the interface betweenthe protein and the DNA, and full use of the data can bemade only if additional information about the threedimensional structure of protein and DNA in the complex

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2217

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Fig. 6. A topological model of the RNA polymerase complexed to a DNA fragment carrying the Al promoter of phage T7 in the closed,intermediate and open complexes. This model was constructed by means of the overall shape parameters of RNA polymerase and DNA obtainedfrom neutron and X-ray solution studies. The details of the structure in the region of the protein-DNA interface based on the results of thefootprinting studies have to be considered as a cartoon.

is available. This structural information at low resolutioncan be provided from neutron and X-ray solution scatteringstudies (Stockel et al., 1980; Meisenberger et al., 1981;Lederer et al., 1986; Heumann et al., 1988). Here we

combine the information provided by these two techniquesand derive a model (Figure 6) displaying the overall structureof the complex.The prominent feature of the DNA protection studies is

the periodic change in the pattern that extends in the closedcomplex over the whole binding domain, and in theintermediate and the open complex over at least two thirdsof the binding domain in the upstream region of thepromoter. This periodicity, along with the observed frameshift of about three bases in the pattern in the two strands,indicates that the major portion of the RNA polymerasemolecule is positioned at one side of the DNA (Tullius andDombroski, 1986). This result is supported by neutron smallangle scattering studies, which have shown that RNApolymerase faces one side of the DNA with an averagedistance between the centre of the RNA polymerase and theDNA axis of (5 + 0.3) nm. Neutron scattering studies havealso demonstrated that the long axis of the RNA polymeraseis aligned parallel to the DNA axis, allowing the elongatedsubunits ,B and ,B' to interact with the DNA (Heumann etal., 1988). The portion of the RNA polymerase that actuallyinteracts with the DNA can be estimated by 'footprinting'studies. Use of hydroxyl radicals as a probe can providedirect information about the size of the DNA domaininteracting with the protein, but only in terms of nucleotidenumbers. The size of the protected area comprises 50 base

pairs in the closed complex and 74 base pairs both in theintermediate and the open complex (Figure 4). If B-DNAconformation is assumed, the length of these sequencescorresponds to 16.5 nm in the closed complex and 24.4 nmin the intermediate and the open complexes. Themaximum size ofRNA polymerase derived by neutron small

angle scattering is (25 h 2) nm. This value corresponds wellwith the estimated size of the binding site on the DNA andshows that the RNA polymerase interacts in the intermediateand the open complexes almost over its whole length withthe DNA. We conclude that the overall structural parametersobtained by our 'footprinting' studies are confirmed by theneutron solution scattering studies.Changes in the 'footprinting pattern' during transition from

the closed to the open complex take place only downstreamof base position -14. These changes at differenttemperatures reflect rather a local alteration in the interfacebetween RNA polymerase and DNA. This view is supportedby previous comparative neutron scattering studies showingthat the structural parameters do not change significantly atthe different temperatures (Lederer et al., 1990).During transition from the closed to the intermediate

complex the protection is extended further downstream by-24 bp. This region of the DNA contains the 'meltingdomain' of the promoter (Metzger et al., 1989). Thisadditional protection suggests that a protein domain in theRNA polymerase wraps around the DNA (Figure 6).The change in the pattern during the transition from the

intermediate to the open complex is restricted to a DNAstretch of nine bases centred around base position -4 only

2218

P.Schickor et al.

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Footprinting of transcription complex

in the codogenic strand. This region within the meltingdomain was fully protected in the intermediate complex andbecomes accessible in the open complex. The temperaturedependence of this accessibility and the formation of aspecific complex (Figure 5) are correlated, indicating that theaccessibility of the bases in the codogenic strand for hydroxylradicals reflects a conformational change in the complex,being important for the formation of a specific complex.Although we have no direct evidence, we suggest that thisconformational change is DNA strand separation, as foundby Kirkegaard et al. (1983). They showed, using the singlestrand specific probe dimethylsulphate, that accessibility ofthe bases at positions -1, -4 and -6 is correlated withunwinding of the DNA. Our studies support this view. Weshow that within the melting domain of the promoter thebases of the codogenic strand only are attacked, in line withprevious findings of Buckle and Buc (1989).The size of the single stranded DNA region, known as

the 'transcription bubble' (von Hippel, 1982), varies between11 bp (as determined by methylation protection of Siebenlist,1979), and 17 bp (Gamper and Hearst, 1982a), as determinedby an unwinding assay of Wang et al. (1977). Accordingto our results, at least nine bases of the codogenic strandshould be involved in the opening process.The consistency of the structural details about the RNA

polymerase -promoter complex obtained by differentmethods encouraged us to speculate about the structuralchanges and the mechanisms involved in the formation ofthe closed, the intermediate and the open complexes. Wesuggested above that contacts of RNA polymerase in theintermediate complex with the melting domain of the DNAbetween base positions -13 and +20 are realized bywrapping of two protein domains of RNA polymerase aroundthe DNA. These 'tube' forming domains (Figure 6) mightbe parts of the subunits ,B and/or ('. Formation of a tube-like structure as the DNA binding site was previouslysuggested from electron microscopic studies on E. coli RNApolymerase (Darst et al., 1989). Whether these conforma-tional changes during transition from the closed to theintermediate complex are facilitated by bending of the DNAtowards the protein, as suggested in Figure 6, is subject tofurther investigation. This hypothesis agrees with theproposal that the DNA within the promoter region is bentin the intermediate complex upon RNA polymerase binding(Heumann et al., 1986; Kuhnke et al., 1987, 1989). Thecentre of the bend is approximately at base position -3(± 20 bp) (Heumann et al., 1989). The bending angle is<45' (Heumann et al., 1988) as illustrated in Figure 6.The question remains as to how the open complex is

formed. We suggest that the protein tube around the DNAis open in the closed complex and closed in the intermediatecomplex. When the open complex is formed, the two DNAstrands are separated within the closed tube, either by activepulling apart of the two DNA strands by means of the twoprotein domains around the DNA, or by sandwiching aprotein domain between the two DNA strands. The strandsare kept separated by that protein domain, which is wrappedaround the non-codogenic strand. This model is in line withthe experimental results showing the non-codogenic strandfully protected and the codogenic strand exposed to thesolution.Opening of the duplex DNA exerts rotational stress on

the DNA duplex in both directions. This stress is at least

partially relieved by rotation of the DNA (Wang et al., 1977;Kirkegaard et al., 1983; Gamper and Hearst, 1982a). Thisrelaxation process requires rotational freedom of the DNAwithin the protected area. The footprinting pattern upstreamof the transcription bubble displays many well defined contactpoints that exclude rotation of the DNA upstream withrespect to RNA polymerase. Due to the stability of theintermediate and the open complexes, association anddissociation processes of RNA polymerase and DNA canbe excluded as a possible stress relaxation mechanism.Therefore, the remaining alternative is relief of the rotationalstress by rotating the DNA downstream of the transcriptionbubble. We speculate that this relief occurs by rotating theDNA within the tube formed by the protein domains ofRNA polymerase suggested above. The protection patternsupports this view. The proposed rotational mobility of theDNA should lead to a variation of the accessibility of theDNA between base positions -3 and +20 along the tube.This is indeed demonstrated by the footprinting pattern ofthe codogenic strand. Downstream of base position +2 theaccessibility decreases until base position + 12 and increasesuntil base position +18 (Figures 3 and 4) in line with areduced diffusibility of the probe into the tube.The proposed mechanism of DNA strand separation, and

the correlated unwinding of the DNA for stress relief,requires some rotational freedom of the DNA within theinteraction site. Flexibility in the interface between proteinand DNA for proper functioning of a protein acting on theDNA is a previously proposed hypothesis (Koudelka et al.,1988; Collis et al., 1989; Metzger et al., 1989).

Materials and methodsPreparation of RNA polymeraseRNA polymerase was prepared according to Zillig et al. (1970). Only thefraction containing core enzyme was used. a-factor was isolated from theoverproducing strain M 5219/pMRG 8 using the method of Gribskow andBurgess (1983). RNA polymerase holoenzyme was reconstituted from coreenzyme and a-factor as previously described (Heumann et al., 1988).

Preparation of DNA fragmentsA 130 bp DNA fragment carrying the T7 Al promoter was preparedaccording to Heumann et al. (1987). The fragment was 5'-end-labelled with[_y-32P]ATP (Amersham) or 3'-end-labelled with [a-32P]dATP (Amersham)according to Maniatis et al. (1982).

Preparation of starter trinucleotide ApUpCApUpC and 32P-labelled p*ApUpC were prepared according to Metzgeret al. (1989).

Preparation of binary and ternary complexesComplexes of RNA polymerase and promoter carrying DNA fragmentswere formed in 8 mM Tris-HCI pH 7.9, 50 mM NaCl and 1 mMmercaptoethanol at temperatures as indicated. The concentration of RNApolymerase was 0.25 mg/ml and that of the DNA 0.025 mg/ml. The volumeof the assay was 20 1I. In order to stabilize the RNA polymerase -promotercomplex, the sample was dialysed against 8 mM Tris-HCI pH 7.9. A10-fold molar excess of heparin was added in order to destroy unspecificcomplexes. Under these low ionic strength conditions the specific complexesremain stably bound, even at low temperature.The complexes were kept at the temperatures as indicated throughout the

whole experiment including the application of the hydroxyl radicals.Temperature constancy is very important due to the fast isomerization ofthe complexes. In our previous footprinting studies (Metzger et al., 1989)incubation of the complexes was performed at 37°C and the hydroxyl radicalfootprinting at 20°C leading to the pattern characteristic of a 20°C complex.Ternary complexes were formed and analysed according to Metzger et al.(1989). The transcription assays were incubated at the desired temperaturefor 40 min.

2219

P.Schickor et al.

Hydroxyl radical footprinting of binary complexesHydroxyl radical footprinting was performed as described by Tullius andDombroski (1986). Since the efficiency of cutting depends on thetemperature, the reaction time for each sample was adjusted to equal cuttingyield.

Purification and analysis of RNA products and hydroxyl radicaldigested DNA on acrylamide gelsThe binary complexes treated by hydroxyl radicals were applied onnon-denaturing gels as described previously (Fried and Crothers, 1981;Gamer and Revzin, 1981) in order to separate the complexes from freeDNA. The complex bands were cut out and eluted as described by Metzgeret al. (1989). After boiling for 3 min the samples were applied on a 10%acrylamide gel as described for sequencing (Maxam and Gilbert, 1977) (7 Murea, 100 mM Tris-Cl, pH 8.6, 84 mM borate, 1 mM EDTA).Electrophoresis was performed at 50 W at a temperature of 60°C.

Temperature gradient gel electrophoresisBinary complexes of RNA polymerase and DNA were formed under thesame conditions as for the footprinting studies, as described above. Incubationof the complexes including all manipulations, were performed at 4°C. Atfirst the complexes were subjected to gel electrophoresis on a vertical gelfor 20 min at 20 V/cm at 4°C in order to allow the complex to enter thegel. Then the gel was transferred to a horizontal temperature gradient plate(Diagen TGGE system) whose temperature gradient reached from 2'C to40°C perpendicular to the applied electric field. After equilibration of thetemperature, the electrophoresis was continued for a further 90 min.

Determination of the boundaries of the protected regionsX-ray films of the electrophoretic pattern of protected and free DNA werescanned on a Hirschmann photodensitometer. Different scans werenormalized by comparing the intensities of bands within non-protectedregions. Boundaries of protected regions were determined by comparingthe absorption scans of digested complexes and free DNA.

AcknowledgementsWe thank Drs F.Pfeifer and M.Horne for valuable discussion andMrs G.Baer for expert technical assistance. This work was supported bythe Deutsche Forschungsgemeinschaft.

References

Kuhnke,G., Fritz,H.J. and Ehring,R. (1987) EMBO J., 6, 507-513.Kuhnke,G., Theres,C., Fritz,H.J. and Ehring,R. (1989) EMBO J., 8,

1247-1255.Lederer,H., May,R.P., Kjems,J.K., Baer,G. and Heumann,H. (1986) Eur.

J. Biochem., 161, 191-196.Lederer,H., May,R. and Heumann,H. (1990) ILL Report 8-03-154, in press.Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) Molecular Cloning. A

Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY.

Maxam,A.M. and Gilbert,W. (1977) Proc. Natl. Acad. Sci. USA, 74,560-564.

Meisenberger,O., Heumann,H. and Pilz,I. (1981) FEBSLet., 123, 22-24.Metzger,W., Schickor,P. and Heumann,H. (1989) EMBO J., 8,2745-2754.

Roe,J.-H., Burgess,R.R. and Record,M.T. (1985) J. Mol. Biol., 184, 441.Rosenberg,S., Kadesch,T.R. and Chamberlin,M.J. (1982) J. Mol. Biol.,

155, 31-51.Schickor,P. (1990) Interaktion der DNA abhangigen RNA Polymerase aus

E.coli mit DNA im binaren und ternaren Komplex, Diploma thesis,University Regensburg, FRG.

Siebenlist,U. (1979) Nature, 279, 651-652.Siebenlist,U. and Gilbert,W. (1980) Proc. Natl. Acad. Sci. USA, 77,

122-126.Siebenlist,U., Simpson,R.B. and Gilbert,W. (1980) Cell, 20, 269-281.Simpson,R.B. (1979) Cell, 18, 277-285.Spassky,A., Kirkegaard,K. and Buc,H. (1985) Biochemistry, 24,

2723 -2731.Stockel,P., May,R., Strell,I., Cejka,Z., Hoppe,W., Heumann,H., Zillig,W.

and Crespi,H.L. (1980) Eur. J. Biochem., 112, 419-423.Straney,D.C. and Crothers,D.M. (1985) Cell, 43, 449-459.Tullius,T.D. and Dombroski,B.A. (1986) Proc. Natl. Acad. Sci. USA, 83,

5469-5473.von Hippel,P.H., Bear,D.G., Winter,R.B. and Berg,O.G. (1982) In

Rodriguez,R.L. and Chamberlin,M.J. (eds), Promoters Structure andFunction. Praeger, New York, pp. 3-31.

Walter,G., Zillig,W., Palm,P. and Fuchs,E. (1967) Eur. J. Biochem., 3,194-201.

Wang,J.C., Jacobsen,J.H. and Saucier,J.-M. (1977) Nucleic Acids Res.,4, 1225-1241.

Zillig,W., Zechel,K. and Halbwachs,H.J. (1970) Hoppe Seyler's Z PhysioLChem., 351, 221-224.

Received on February 2, 1990; revised on April 10, 1990

Amouyal,M. and Buc,H. (1987) J. Mol. Biol., 195, 795-808.Buc,H. and McClure,W.R. (1985) Biochemistry, 24, 2712-2723.Buckle,M. and Buc,H. (1989) Biochemistry, 28, 4388-4396.Chamberlin,M.J. (1976) In Chamberlin,M.J. and Losick,R. (eds), RNA

Polymerase. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,NY, pp. 17-67.

Collis,Ch.M., Molloy,P.L., Both,G.W. and Drew,H.R. (1989) NucleicAcids Res., 17, 9447-9468.

Darst,S.A., Kubalek,E.W. and Kornberg,R.D. (1989) Nature, 340,730-732.

Fried,M.G. and Crothers,D.M. (1981) Nucleic Acids Res., 9, 6505-6525.Gamper,H.B. and Hearst,J.E. (1982a) Cell, 29, 81-90.Gamper,H.B. and Hearst,J.E. (1982b) Cold Spring Harbor Symp. Quant.

Biol., 47, 455-461.Garner,M. and Revzin,A. (1981) Nucleic Acids Res., 9, 3047-3060.Gribskow,M. and Burgess,R.R. (1983) Gene, 26, 109-118.Hawley,D.K., Malan,T.P., Mulligan,M.E. and McClure,W.R. (1982) In

Rodriguez,R.B. and Chamberlin,M. (eds), Promoters Structure andFunction. Praeger, New York, pp. 54-68.

Heumann,H., MetzgerW. and Niehorster,M. (1986) Eur. J. Biochem., 158,575-579.

Heumann,H., Lederer,H., Kammerer,W., Palm,P., Metzger,W. andBaer,G. (1987) Biochim. Biophys. Acta, 909, 126-132.

Heumann,H., Ricchetti,M. and Werel,W. (1988) EMBO J., 7, 4379-4381.Heumann,H., Lederer,H., Baer,G., May,R.P., Kjems,J.K. and Crespi,H.L.

(1989) J. Mol. Biol., 201, 115-125.Hofer,B., Miller,D. and Koster,H. (1985) Nucleic Acids Res., 13,

5995-6013.Kirkegaard,K., Buc,H., Spassky,A. and Wang,J.C. (1983) Proc. Natl. Acad.

Sci. USA, 80, 2544-2548.Koudelka,G.B., Harbury,P. and Ptashne,M. (1988) Proc. Natl. Acad. Sci.

USA, 85, 4633-4637.

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