,I. Mol. Riol. (1991) 219, 201-215

Catabolite Activator Protein-induced DNA Bending in Transcription Initiation Sandra S. Zinkel and Donald M. Crothers Departments

of Molecular Biophysics and Biochemistry and Chemistry Yale University, New Haven, Cl’ 06511, U.S.A.

(Received

11 July

1990; accepted 14 January

1991)

We describe experiments that enable us to track the presence and direction of the DNA bend induced by Escherichia coli catabolite activator protein (CAP) through the intermediate stages of transcription initiation at the lac promoter. Transcriptional complexes examined were formed on superhelical templates to enhance specific complex formation, and detected by electrophoretic analysis after restriction digestion. We found that the bend is maintained and even increased upon formation of closed and open complexes. Our results exclude the hypothesis that the energy of the CAP-induced bend is used to promote open complex formation. We now suggest a new model, in which DKA wraps around the CAP-polymerase complex to form a writhing structure equivalent to that at the end of an interwound superhelical domain. Formation of this structure may facilitate open complex formation. We further propose that the stored bend energy may be used to help counteract strong protein-protein or protein-DNA interactions, thus assisting the process of RNA polymerase escape from the promoter. Keywords:

CAP/CRP protein; RNA polymerase; open complex; DNA bending; transcription initiation

1. Introduction Regulation of transcription initiation in both prokaryotes and eukaryotes is mediated by DNA-binding proteins that attach upstream fromthe transcription start site and either repress or activate transcription of the adjacent gene. Despite the prevalence of proteins in regulatory regions that bend DNA upon binding, a mechanistic role for this distortion of DNA structure remains elusive. The catabolite activator protein (CAP?) from Escherichia coli, t,he prototype of a Dh’A-bending regulatory protein, stimulates transcription of the genes encoding proteins involved in lactose metabolism. In t,he presence of CAMP, CAP binds to a site upstream from RNA polymerase and stimulates transcription approximately 50-fold. Two classes of models for CAP stimulation have been proposed: one involving protein-protein interactions (Gilbert, 1975), and one involving changes in DNA struct,ure between the CAP and RNA polymerase binding sites (Dickson et al., 1975; Wartel, 1977; Weber & Steitz, 1984). There is as yet no

decisive evidence that eliminates either model as a contributing factor in transcription activation. Upon binding to DNA, CAP protein induces a substantial bend (Wu & Crothers, 1984), estimated by gel measurements to be 100” to 130” (Thompson & Landy, 1988; Zinkel & Crothers, 1990). Calculation of the free energy required to produce a similar DNA conformation (Liu-Johnson et aE., 1986) suggests that such a bend would occur spontaneously due to the thermal flexibility of DNA with a probability of less than 10m6. It seems plausible that this stored free energy may be released during transcription initiation and used to perform mechanical work, but it is not known how such a transduction might be accomplished. According to the model of transcription initiation proposed by Chamberlin (1974), RNA polymerase (R) first binds to the promoter (P) and forms a closed complex (RP,), which is competitor sensitive. The polymerase then isomerases to an open complex (RP,), which is competitor-resistant and in which 12 bp of DNA are unwound (Kirkegaard et al., 1983; Siebenlist et al., 1980). Addition of rNTPs produces a stably initiating complex with the loss of sigma

t Abbreviations used: CAP, catabolite activator protrin: hp. base-pair(s): knX;A. kinetoplast DKA.

tactor (Carpousis & Gralla, 1985; Straney Crothers, 1985). Each of these intermediates

&

in

201 oo22-2s3s/~I/Itn20115

$03.00!0

0 1991 Academic Press Limited

A’. S. Zinkel

nnd 1). M.

transcription initiation can he isolated as a protein-DNA complex on a polyacrylamide gel (St)raney & G-others, 1985). Measurement of the kinetic parameters for wildtype (wt,) lac DiVA promoter fragments, compared to a promoter into which a 5 bp segment of Dh’A was inserted hetween the C!AP and RNA polyrnerase sites, indicates that CAP increases the initial binding constant. K,, hy approximately threefold, and increases the forward isomerization rate. k,. hi a larger factor. There is no detectable effect on thP rate of formation of the initiated complex from t,he opctn complex (Stjraney et nl., 1989). To address the quest’ion of the role of the (!AP bend in the biological function of the protein, ue monitored the (‘AP-induced hend during transcription init’iation using a grl elec%rophoresis assay to separate the transient protein-11X.4 complexes. and to detect, small bends in t,he DNA (Strant>y & (brothers, 1985; Zinkel 8 (brothers. 1987). The assa) uses a DNA substrate in which a sequence-directed bend from the kinetoplast DNA (kIlNA) of L&hmania tarrntolaP is placed next to the repulwtory region of the luc operon. and thr phasing hetween the sites is varied over one helical t’urn by addition of linkers (Zinkel & (‘rothers, 1987). The sequence-induced bend has the effect of amplifying small changes in DNA bending at the sec~md site 1)~ magnicying changes in the end-to-end dist,anec. upon which gel mobility depends in a non-lineal manner (Lerman 8 Frisch, 1982: Lumpkin B Zimrn. 1982: Koo 8: (It-others, 1988). For reasons descarihrd hy Zinkel (1989) and Gartenherg rlt al. (1990). MY now prefer the phase-dependent assay over the easier and more commonly used circular prrmutat,ion assay (\Vu & (‘rothrrs, 1984) in caases wherrh the protein makes the dominant c~ontrihution to t hr frictional effects in the gel. Our experiments depart from earlier approaches. in that the promoters studied arc contained in superhelical DNAs rather than in restriction fragments; restriction digestion subsequent t)o caomplrx formation allows assay hy standard non-drnatlrring polyacrylamide gel electrophoresis. with detection hy ethidium fluorescence. The presence of mult,iple DNA bands on t,he gels did, however, significantly idenGfying the problem of the complicatt complexes. We adopted this strategy in order to take advantage of the ability of superhelical tension to increase the yield of complexes specific> to lrcr promot,er.

2. Experimental

(‘rothrrs

Sequence of kPGem 140/l RSOI ACATATTG;: TGTATAACAG

GTTAGAACG:’ CAATCTTGCG

CTT ATGTA% GAATACATAG

ATACACATAC TATGTGTATG

GGCTACAA% CCGATGlTAA

60

EC0RI90

70

140

CAAAAATCCC GTT TTTAGGG

150

AAACmTA TITGAAAAAT

CCGGAAGCTT CCGGCAAGCTTG

sad

GAGCTCGC CTCGAGCG

200

210

TTAATGTGAG AATTACACTC

BstN1240

CCCAGGCT TT GGGTCCGAAA mJWAN280

cc CC

CAPAh TTA GCTCACT AATCGAGTGA

250

ACACTT TATG TGTGAAATAC

320

TATGACCATG ATACTGGTAC

230

CATTAGGCAC GTAATCCGTG 270

GTATGTTGTG CATACAACAC

300

Ah1

310

AGGAAACAGC T CCT? TGTCC

HhfI330 ATTACGGATT TAATGCCTAA

&mHI 350 GCGGATCCTC CGCCTAGGAG

PSlI

370

CTGCAGCCCA GACGTCGGGT

400

GTGAGTCGTA CACTCAGCAT

&7/nH1190 GGGGATCCGC CCCC TAGGCG

AGCGGATAAC AAmCACAC TCGCCTAT TG TTAAAGTGTG

360

TAGAGTCGAC ATCTCAGC TG

160

M+pI260

CT TCCGGC TC GAAGGCCGAG

repressor290

TGGAATTGTG ACCTTAACAC

EcaRI

GGTGGAATTC CCACCTTAAC

180

CCGGACAAGCTTGT CCGGCACAAGCTTGTG CCGGTCACAAGCTTGTGA CCGGAAGCTTCCGGAAGCT

CGCAACGCAA GCGTTGCG’M

80

ACACTATAGA TGTGATATCT

110 Bend cmtre CAAAAATGTC AAAAAATAGG GTTM T ACAG TTT TI-TATCC

l-l-C CAATTCC AAGGTTAAGG

130

CAAAAAATGC GTT TT TTACG

HPhI

GA’MTAGGTG CTAAATCCI’C 100

ATACACGGAA TAT GTGCCTT

AATACAT A: ‘ITATGTA T-I-G

340

CACAGGAATT GTGTCC TTAA HindllI 380 AAGCTTCCGG TTCGAAGGCC

410

TTAAmCGA AATTAAAGCT

390

TCT CCC TATA AGAGGGATAT

PVUII

TAAGCCAG ATTCGGTC

of the DKA constructs. The Figure 1. Seyuenw sequence is shown from t,hr /ZsoT sit,? of t,hr I+uII site of the parvttt plasmid (p(4etn2. I’romrga). A fragment of kinetoplast DSA (kl)N‘A) ttas hren inserted into tlrtb

/CUJRT sitr of thta plasmid. linktars varying in length from IO to 20 bp bare been inserted into the rll~]I sit?, and a portion of the /UC protnot,rr rrgion has been insert4 into the Ha~rHl site sucah that proximal to thr kl)XA bend.

RXA

polymerase

the

holoenzymc

(‘AI

(kindly

binding

site

providrd

is

t)>

I>. C’. Straney and S. 13. Straney) was puri’fied from E. roli KI 2 cells (Grain Processing Corp.) using the proccbdure ot Burgess C ,Jendrisak (19’75) and then Gonzales et al. (1977) to obtain holoenz?;me frer of contaminating c-ore r~rtz,vtnr. ((4) (‘.-II’

(‘AI’ protein (kindly provided by I)r A. I%rowtr) wax purified using an affinity chroma,tography trchniquch (Brown, 1987).

Procedures

(a) DIVA The sequences of the D84 samples used are given in Fig. I. Plasmids were isolated according to the method of Clewell & Helinski (1969) and ClewelI (1972). An addtional precipitation in polyethylene glycol (654b (w/v) in 02 M-NaCl) was performed to remove any contaminating RKA before forming transcription complexes.

RNA

polymerase-

(‘AFI)NA

complexes

werp typically

formed by caombining I pg of plasmid I)R;A and 0.125 /~g of CAP in 10 rnbr-Hrprs (pH 8.0). 0-I tn.~-dithiothreitol. 0.1 mnt-EDTA. KS tnM-potassium glutamatr 100 ~ib-~.AYl I’. 0.5 mp bovine serutn albumitt]tnl reaction volume. Thr reac+ons wert’ incubated

(pH 8.0). in a 3O-~I at room

temperature (20°C:) for 15 min. For closed complex formation. MgCl, was added to I5 rnM and the r’pactions wc’rc’

CAP-induced brought to 16°C before t,he addition of 025 pg of RNA polymerase holoenzyme. The reactions were incubated for 2 to 5 min at 16°C. Ten units of RsaI and 10 units of f+uTI (Sew England Biolabs) were added and the reactions were incubated for an additional 30 min. Dye buffer (lOxbuffer:60’& blue. (w/v) sucrose, bromophenol xylene csyanol) at 16’(’ was then added and the reactions were loaded immediately ont,o a 3.2% (w/v) (ackrylamide to his. 39 : 1. M./W) 8!) mw-Tris-borate (pH 8.4). 2.5 mM-El)T;\ gel (P) Open, romplexes CAP -I>xA complexes were formed as for closed caomplexes, with the exception that 0.375 pg of CAP were added. For open complex format,ion. MgCI, was added to I5 ITIM and the reactions were brought to 37°C. after which 0.75 pg of RNA polymerase holoenzyme were added, and the reactions were incubated at 37°C for I2 min. Dye buffer and cotnpetitor at 37°C: were then added and the reactions werr loaded onto a native polyacrylamide gel. which was run at either 2OY” or 37°C’. (!omprt.itor: I pg of a 32 bp duplex oligomer containing 3 A-tracts in phase wit,h the helix sqrew. or 80 fig heparini ml. sodium salt (Sigma) as noted.

DNA Bending

203

closrd and open complexes, the gels were stained with ethidium bromide and visualized by ultraviolet light on a transilluminator. Gels were photographed with a Polaroid camera. In the case of initiated complexes, the complexes were visualized by autoradiography on DuPont Cronex film at 4°C. (h)

Idrntijkation

Initia,tetl

complexes

Open complexes were formed as above. For the formation of initiated complexes, concentrations were adjusted to 50 PM of the dinucleotide GpA (Pharmacia) and 10 PM each of 3 nucleoside triphosphates (ATP, GTP and UTP; Pharmacia). The 4th nucleoside triphosphate (CTP) was omitted in order to stop transcription at the point of incorporation of the 1st CTP, which is the 11th base of the RNA chain. Initiated complexes were generally labelled by means of the mRI%A by the addition of 50 FCi of [rx-32P]ATP (Amersham) in the NTP mix. The reactions were incubated at 37°C for 12 min. Dye buffer and competitor at 37°C were then added and the reactions were loaded on a gel as above. In the case where the complexes were labelled with radioactive NTPs recirculation of the running buffer was stopped just before the point where the free label entered the bottom buffer (20 min). The concentration of (*AMP in the top buffer was then maintained by the addition of cAMP to 10 jLM at 20 min intervals during the course of the gel run. Competitor: heparin, 80 &nl, sodium salt (Sigma). Measurements of gel mobility werp made from the films. In some cases (Fig. 7(a)), the complex gels were stained with ethidium bromide after autoradiography. The autoradiogram was then placed over the gel on an ultraviolet light transilluminator to determine which bands were radiolabelled. This direct comparison was necessary since the lac initiated complexes sometimes comigrated with other. irreproducible. bands on thch gel. These latter were not csharactrrized further. (g) Uel elecfrophoresis

Reactions w(lrt’ loaded onto a 47” TBE gel (89 mm-Tris-borate (pH 8.4). 2.5 rnM-EDTA: 39 : 1 (acrylamide t,o bis. 39 : I. w/w) at 200 V. Once the reactions entered the gel. the voltage was increased to 450 V. The running buffer was TBE+ 10 PM-CAMP and was recirculated to maintain a steady concentration of cAMP in the gel. The temperat)urr was maintained to within 1 deg.(’ with a constant-temperature gel apparatus (Hoeffer Scientific). Gels were run until the xylene cyanol was within I cm of the bottom of a 16 cm gel, In the case of

RNA

transcripts

TO analyse the RNA content of the initiated complexes, the initiated gel band was excised from the gel. treated with 15 ~1 of 7 ,vn-urea, 1 y/, (w/v) SDS, bromophenol blue, and lrft at room temperature for 2 to 12 h. The gel slice was inserted into a 1.5 mm thick 20% polyacrylamide (20 : I (w/w) acrylamide to bis). 7 M-urea, TBE gel. The R?iA was electrophoresed at 75 W (hot to the touch) until the bromophenol blue dye had travelled 23 cm and then autoradiographed at -70°C. The size standard was a mixture of synthetic DNA oligomers (lengths of 10, 12, 14. 16. 18. 22, 28 and 36) that had been radioactively labelled with [y-32P]ATP and polynucleotide kinase. A parallel reaction was always run with a linear 203 bp pitLc*caof DNA fragment containing the wild-type Zac promotfsr. (i)

if)

of

Western

blots

Open complexes were run as above in the presence of heparin as a competitor, which yields 2 open complex bands (see Results). The gels were stained in a higher concentration of ethidium bromide for 10 min and photographed as above. Gel slices containing the protein-DNA complexes of interest were excised and placed in soaking buffer (375 mi\l-Tris. HCI (pH 7.5), 1% SDS) for 1 to 2 h to negatively charge the CAP protein. The Western blot protocol followed the procedure of Granger-Schnarr et ul. (1988). Protein was blotted to PVDF membrane (Milliporv) for 2 h at 85 V (250 mA). The blot,ting was done at 4°C’ in a Bio-Rad blotting apparatus in .5 I of 20 mm-Tris. I50 rnM-glycine, 2Oo/o (v/v) methanol. After blotting. the filter was blocked for 1 h at, room temperature in filtered blocking buffer (0.9% (w/v) ?u’aCl. 20 mM-Tris. HCI (pH 7.4). 5S;, (w/v) bovine serum albumin). The filter was inc*ubatecl overnight with gentle agitation at room temperature (20°C) in 20 ml of blocking buffer containing 0.1 Ob (v/v) Triton X-100 into which CAP-specific monoclonal antibody mAb 64B4 (Li & Krakow. 1985) was diluted l/1000: the monoclonal antibody was a generous gift from Dr Joseph Krakow. The antibody was decanted, and the filter was rinsed 4 times for 5 min in TBS (O.Y%, Kat(‘l. 20 mhl-Tris, HCl (pH 7.4)). tk 1% Triton X-100. The membrane was blocked in blocking buffer for 30 min. then incubated with the 2nd antibody (goat anti-mouse IgG alkaline phosphatase conjugate; Sigma) at a l/l000 dilution for 2 h. The filter was washed once for 15 to 20 min and twice for 5 to 10 min with TBS containing O~l’~,~ bovine serum albumin. The phosphatase activity erosslinked t.o t,he anti-antibody was revealed using 0.14 mM-5-bromo-4-chloro-3-indolyl phosphate (Sigma), 1 rn~MgCl, in 100 mM-diethanolamine buffer (pH 9-8). Staining was followed by eye and stopped after a few minutes by rinsing in doubly distilled water when no further increase in band intensity was seen. The filter was allow-ed to dry in the dark. as the stain is light sensitive. (j)

Measurement of the relative of the complexes

mobility

The mobility of the complexes was measured from the bot,tom of the well of the gel on a Polaroid photograph or

autoradiogram of the gel. C’omplex mobilities M~‘IY’ c:orrec%ed fbr the distance migrated into the gel. and ~CJI, the variation in gel mobility due to that of the free I)NA\ as described earlier (Zinkrl 8: (‘rothers, 19X7). In thus ,‘itst’ of the c:losed complexes. it was necarssary also to c~orr’ec~t for the decrease in variation caausetl by running the. c*omplexes on a 39 ‘lo rather than a 1-O,, gel. This (Y)I’I’CYtion was done by fitting the plots for thr (‘Al’ 1)S.t c*omplex on both X?(~, and 4’j(, grls to x siw wrw~ usittg a least-squares program based on a c~onibination ol’ the and the m&hod of’ steeprst Maryuardt algorithm descents. A ratio of the amplitude of the 4”;, (‘urv(’ to the amplitude of the XZ”;, curve was c~alculated to be IN. The data points for the 3.%“,, grel were c~orrrc+31 by the equation (I)- I) (1.!)4) + I. (I) = thrb relat.ive mobilit!~ 01 f’ value for the X.2’),, plot .)

We estimate the fractional uncertainty in t’he measunment of gel tnobilit,ies to be approximately one-half of a band width (00.5 cm) divided by the total tlist,anc’e tnigrat,ed. The error for t.hr open c~otnplrxes that &tained (‘AI’ was slightly higher than t,hat for the other complexes studied due to the smaller migration into ttw gel and the larger band widths obtained on autoradiograms. It should be noted that the plotted data points are t,hr result of averaging the values obtained from 6 indrpendent, experiments in the case of open complexes. and from 4 to 6 independent experiments in the cast of initiated complexes. The maximum standard deviation for any value of relative mobility was Zoo: t.he estirnatc~tl

3. Results

complexes on DNA rrstricat ion fragments. fi)llowitiy the general approacah of’ Stranry & (‘rot ht>rs ( 1985). FVe found that the weakness of the lag prorrrot~~r otl linear templates allowed substantial c~ompf~titiori from an unantieipat,ed sec.ontl promoter, c7eat.t:tl in the molecules containing a sl’ciueri(.e-(iir.rc.~(‘~l ht~ntl. The results of those experiments remain of interest because they povidr a c.ontrol that vrrifitas the sensitivity of the elretrophoresis assay ti)r thcl tlrt,ec~~ t,ion of (‘APinduced I)NA hrnds in then prrsrnc’r ot bound RNA polymera.se. As out,lined in Figuw :!, we inc~ubwted lmrifircl (‘AT’ protein and RNA polyrnerase with a serif3 of DNA restric:tion fragments that l~arl~ollrrtl il srqu~‘“(‘e-dire~t,e~l bend f’iwm thr kitlrt(,plast 1)N.A of L. tnwntolor. phased over oncb helic~al turn relwlivc, to the regulatory region of t,he 10~ optlron. Our experiments showed that E. roli RNA polymerast~ binds strongly to two dit?‘erent sit,es on I hesr linear templates: at t,he 1~ promoter. and at a11 additional Linear

DNA

al

0 RNAP

0

RNAP

I

co t

CAP

CAP t rNTPs

RestrIction endonucleases

t rNTPs

&WI

t

PvurI

--

i

v

SupercoOed plasmid Low moblllty High moblllty

-

-

Polyacrylamlde gel

Figure

2. Design of the experiment. Protein-DNA caomplexes were formed by incubating super(*oiled 1)X1\ with (“Al’ and RNA polymerase as described in Experimental Procedures. After complex formation. the plasrnid 1)X,4 was cut with KsaI and PvuII to obtain DKA fragments of a convenient size and run on a native polyac.rylamidr gel. The complexes were visualized either by staining with ethidium bromide, or in the case of initiated complexes. b>. invoqmrating [cc-~~P]IJTI’ into the transcript. thereby radiolabelling the complex by means of the RN.4. I)klrified

CA P-induced DNA Bending 1

I- /-.- i

205

undetermined site. Transcription from the alternate site depended both on sequences upstream from the kDNA insert, and on sequences in the ZUCpromoter up to and including the CAP binding site. but not the -35 or - 10 regions. These transcripts were not stimulated by (:AP. Initiated complexes on the alternate promot’er were identified by their dependenvr on the presence of rXTPs in t,he reaction mixture. and their production of transcripts that did not correspond to the expected transcripts from the Inr promoter (as determined by radiolabelling t,he cvmplexes with rXTPs, and running them on a gel cvntaining %09,, urea: dat’a not shown). The gel slices containing t,he complexes wercb also run on a polyacrylamide/SDS

gel to verify

the presence

of

RNA polymerase (data not shown). A plot, of t)he relative non-denaturing gel mobility zwsus linker length for t’hese initiated complexes

I I IO

III 12 14

16

I I 18 20

II IO

III 12 14

16

I I 18 20

Lmkw length (bp)

Figure 3. 1’lot.s of relative mobility versus linker 1engt.h. (a) nosed complexes. Measurements of closed complexes were made on a 3.:!‘?, gel, and scaled to measurements of (‘AI’ complexes on a 4% gel (see Experimental Procedures). (+) (141 complexes; (w ) closed complexes. ‘I’he variation in gel mobility due to phasing for the closed complex is approx. l/3 of the smplitude of the CAP caomplrx. (b) (‘ompositc graph of all of the complexes. (+) (‘AI’ cSomplrx: (m) closed complex: (A) open complrx - (‘AI’; (v) open complex + CAP; (0) initiated complex. (c) and (d) Open romplexes. (c) The complexes wertb formed at’ 37°C‘ and run on a gel at 37’C. (d) The c*ort~plrxes were formed at, 37 “(: and run on a gel at 20°C. (+) (‘Al’ complexes: (A) open complexes - CAP; (V) open complrxrs + (YAP. The phase-dependent gel mobilit?- variat,ion for open complexes - CAP is approx. l/3 of thr amplitude and in phase with that, of the CAP ~omplrx. The phase-dependent gel mobility for open complexes + (‘.\I’ is equal in amplitude to that of the open complrx - (‘Al’. but shifted in phase bg 180”. (e) and (f) Tnit.iatrd complexes. (e) The complexes were formed at 37°C’ and run on a gel at 37°C. (f) The complexrs werp formed at 37°C: and run on a gel at 20°C. (+) (‘AI’ complt~xrs: (A) lower initiated complexes; (0) middle initiated complexes: (m) upper initiated complexes. The initiated complexes show little phasedependent varia.tion in gel mobility at 37°C. This variation is significantly enhanced at, 20°C for all complexes. and is t&specially prominent for the 16. 18 and 20 bp linker tengths. (g) Protein-DNA complexes were formed on linear I)IVA. (+) CAP-DR‘A complexes; (0) initiated complexes on alternate promoter; ( n ) initiated complexes on alternate promoter + heparin. The phase-dependent variation in gel mobility for the alternate initiated complex is approximately equal to that of CAP, but decreases to background upon the removal of CAP protein with heparin. (h) (‘AP-DNA complexes phased against 3. 4 and 5 A tracts. The molecules contained a 216 bp extension of straight DlVA beyond the centre of the CAP bend. (A) Three .4 t,racts; (0) kDIVA (4 A tracts); (m) 5

(Fig. 3(g), where a comparison is made to the same plot for (‘AT’ complexes) indicates t.hat the phasing effecat on gel mobility, a function of bend magnitude and direction, is nearly equal to that of CAP bound alone. Li’hen CAP was competed from the initiated complexes with heparin, the absolute mobi1it.y increased

dramatically,

and the phasing

decreased

to the Itvel of background, indicating that the bend had disappeared. Hence, in the presence of independently bound C\P and RNA polymerase. t,he gel assa)’ is able to detect protein-induced I>;I’A bends. In addition, the apparent magnit,ude of the bend. as judged

from

the

peak-to-peak

amplitude

of the

phasr-dependent to hurd by binding

variation of relative mobility due (:A1 (Fig. 3(g)), seems not to be affected of the large FtSL4 polymrrase

molrculr.

Once the sensitivity of the method was &ahlished. we fovnsed our attent.ion on enhancing the yield of‘ complexes that originated at the lnc promoter. Lf’e adopted the strategy of forming the protein-I)NA complexes on supercoiled DNA. since supt~rhelical

st)ress

increases

the

rat,c

of

op(?n

complex formation from the lar promoter SO-fold (f3orowiec & Graila, 198.5). This increase in the rate of open ctomplrx formation allowed the Inc promoter to vompetr effective!? for RNA polymerase with the unidentified

adventitious

promoter.

After

complex

formation, the plasmid DNA was CYI to a (bonvenirnt size (approx, 130 bp) with t.he appropriate restriction enzymes and run ori a native polyacrylamide gel (Fig. 2). The complexes were visualized eit hf>r 1)~ staining with et,hidium bromide. or in the cast’ of initiated ILY-~‘P]~TT into

labelling (‘omplrxes

complexes, by incorporating the transcript, thereby radio-

the complex werf’ assigned

by means of the to the Inr promoter

.1 tracts. The largest amplitudr modulation in gel mobility c*ont;tining 5 A tracsts.

RN\;X. on the

in the phasr-dependent occurs for molecules

basis of their (1A I’ stimulation. and thrir ability to produce transcripts corresponding in sizcbto t hohCxof the wiltl-type lrtr promoter fragment under similar assay c~onditions. In t’he case of open complexes. t hfa presence of CAP was also verified i)y a LYestern blot of the protein-l)NA complexes. Tt should he ernph:~sized that). although the complexes are formcltl on superhelical IINA. the I)SA is linear at t.hc time of the assay. This facailitat)es analysis of bends in the l)SX. but’ does not allow us to draw Wncalusiotis concrrning para,meters that are relevant only to Grcular l)NA, such as linking number. (c) Identijication of initiated complexes the lac promoter

initiated

properties complex

qf the

RNA-polymerase-lac initiated Once the promoter complexes were assigned. we formed initiated complexes on substrates with altered phasing between the promoter and the kDNA bend. It should be noted that in these reactions, heparin is present as a competitor that removes CAP protein from the initiated complex (Straney et al., 1989). These complexes were run adjacent to CAP-DNA complexes formed on the end-labelled, linear DNA (Fig. 5) to allow a correction of the gel migration due to variations in the mobility of the free DNA as well as to normalize any change in gel mobility due to spurious inhomogeneities in the electric field of

b 2ooc 3 -

CAP lJ+ lac

1 c- IOC /ac

+--L

[ L-

NonIOC

4 +

f-u f-M

M-

Non-

--)

(0)

at

Initiated complexes were the simplest to identify, because they can be distinguished by their ability to be radiolabelled upon addition of [cc-~‘P]NTPs to t.he reaction mixture. Initiated complexes that originated at the lac promoter were identified on the basis of stimulation by CAP (Fig. 4(a)) and characterization of the transcript extracted from the nondenaturing gel slice. The lengths of the RXA chains were determined by direct comparison (on a 209; denaturing gel) to transcripts formed by t.he wildtype 203 bp Eat promoter fragment under identical conditions. Only three nucleoside triphosphates were added in order to obtain primarily transcripts that terminated before the positions of CTP incorporation. Three initiated complexes of differing elect.rophoretic mobility were identified on the basis of C’AP-stimulation and transcript length as having originated at the Zac promoter; no CAP-stimulated complexegwere seen on the control plasmid (which has no lac sequences) under these conditions (data not shown). The initiated lac promoter complexes. from highest, to lowest m0bilit.y. contained predominantly transcripts of 10, 17 and 23 nucleotides in length, consistent with pausing at the positions in the transcript where CTP is incorporated. The two larger transcripts presumably arise either from misincorporation of other nucleotides. or from thP presence of a small amount of contaminating CTP in the nucleotide mixes. (d) Electrophoretic

a

lot

Iac

-23

(b)

Figure 4. CAP-stimulation of initiated complexes. (a) Initiated complex gels: a, initiated complexes were formed on the construct that contained the 12 hp linker as described in Experimental Procedures, and the reactions were run on a 4% TBE gel at 37°C; b. the reactions from a were run on a 4% TBE gel at 20°C. Three complexes show a significant CAP-stimulation. (b) Transcript)s from initiated complexes. Initiated complexes were excised from the gels shown in Fig. 3(i), soaked in urea buffer. and loaded onto a denaturing gel. Transcript lengths were compared to those produced under identical condit,ions on a fragment of DKA containing the wild-type lac promoter region (wt203). Lane 1, lower non-lac complex; lane 2. upper non&c complex; lane 3. lower lac complex (predominantly 10 nucleotide transcript); lane 4, middle Zac complex (predominantly 17 nucleotide transcript); lane .5. 23 nurleot,ide (predominantly upper lac complex transcript).

the gel. A plot of the relative gel mobility cersus linker length for the initiated complexes at 37°C and 20°C is shown in Figure 3(e) and (f). At 37°C. there

is very

little

phase-dependent

modulation

in

gel mobility. However. at 20°C. the complexes that contain the longer transcripts show a phasethat of gel mobility modulation dependent increases. while the absolute gel mobility of these

CAP-induced

I

2

3 Labelled

4

5 NTPS

DNA

6

207

Bending

7

6

9 Labelled

IO

II

12

DNA

-

CAP

C-

Free DNA

Figure 5. Initiated complexes. Initiated romplexes were lahelled with [cr-32P]UTP and run on a gel at 37°C next to (YAP-DXA complexes. Lanes 1 to 6. initiated complexes on DE;A constructs containing linker lengths from 20 to 10 bp. respectiveI>;: lanes 7 to 12, CAP-DEA complexes were formed on linear DFA that was labelled with [Y-~~P)ATP and centainrd lmker lengths from 20 to 10 bp. respectively

complexes decreases with transcript length. These data suggest that as the polymerase moves down the DXA, it gradually adopts a less compact structure and therefore has a slower gel mobility. This may result from a change in the structure of the polymerase, from the increasing frictional effect of the transcript, or from the formation of weak interactions with the DNA that produce a bent structure.

(e) IdentiJication

of open complexes

To identify open complexes, ternary CAP-DKA polymerase-D?iA complexes were formed in the absence of nucleotide triphosphates and visualized by staining the polyacrylamide gels with ethidium bromide. Two complexes appear that were both competitor-resistant and CAP-dependent. Althou’gh both complexes were clearly distinguishable at 37 Y! of the lower (Fig. 6(a)), at 2O”C, the identity complex was obscured due to the comigration of several bands that were not specific to the lac operon. The Eat-specific complexes were unstable to high salt (O-35 M-KCl, data not shown), and were chased into initiated complexes by the addition of nucleoside triphosphates (Fig. 7(a)), consistent with the behaviour of lac operon complexes (Straney & Crothers, 1985). On the basis of their resistance to competitor, their CAP and lac promoterdependence, and their documented role as precur-

sors to initiated complexes, we defined these complexes to be lac promoter open complexes. The upper open complex can be shifted into the lower open complex by titration with heparin, which has been shown to remove CAP protein from DNA (Straney & &others, 1987aJ). In Western blots, CAP was apparent only in the upper open complex (Fig. S), although the formation of both complexes was CAP-dependent (Fig. 6(a)a, lane 2). Only the upper open complex appears in the presence of a DNA competitor that dissociates RNA polymerase closed complexes but is unable to remove CAP protein (Figure 5(a) lanes 5 to 10). The only simple explanation of these data is that CAP is lost from the open complex at high concentrations of heparin, leaving a second open complex of higher mobility. (f) Electrophoretic

properties

of the open complexes

The two open complexes have distinctive electrophoretic properties, both in mobility and in their phase dependence. As detailed in Discussion, the relatively large magnitude of the difference in gel mobility between the two complexes is consistent with expectation for loss of the CAP-induced bend when CAP is dissociated from the CAPpolymerase-promoter open complex; the effect is too large to be explained by loss of CAP without change in bending. A plot of the modulation in open complex gel

208

S. 5’. Zinlcel and I). M. (‘rothwrs

’ I

2

3

4

5

67’6

Figure 6. Typical gels. (a.) Open complexes at 37°C”. Prot.ein-DNA complexes were formed at 37 “C as described in Experimental Procedures. Either a 32 bp duplex oligomer containing 3 A tracts or heparin (80 pg/ml, only in lane I ) was added to the reactions as a competitor immediately before loading on a gel (the oligomer competes efficiently for nonspecifically bound polymerse. but does not compete for CAP. Heparin removes CAP from all but the open complex). a. Lane 1, complexes formed on DKA containing the 12 bp linker + heparin; lane 2, complexes formed on D2iA caontaining the 12 bp linker - CAP; lane 3. complexes formed on control DNA (no lac promoter) + C’AP: lane 1. complexes formed on control DBA - CAP; lanes 5 to 10. complexes formed on 20 to 10 bp linker DiXA, respectively. h. IAonger migration of Protein-DKA complexes. Lanes f to 6. complexes formed on DNA containing 10 to 20 bp linkers. respectively; lane 7. control DR;A - CAP. (b) Closed complexes. Protein-DXA complexes were formed at 16”(! as described in Experimental Procedures, and run on a 3.20/,, gel. Lanes 1 to 6, ceomplexes formed on DlrjA containing 10 to 20 bp linkers. respectively. Lane 7. complexes formed on DXA containing a 12 bp linker - (!AP. Lane 8. complexes formed on control D,h;A + CAP.

mobility due to phasing is shown in Figure 3(c) and (d). In the case of the lower open complex (-CAP), t’he variation was in phase with that of the CAP bend in other complexes. However, the upper complex, ( + CAP), showed a small but reproducible, phase-dependent variation in gel migration that was shifted 180” (approx. 5 bp) with respect to CAP. We defer interpretation of this result to the Discussion. On our substrates, the CAP molecule in open complex was much less stable in the presence of heparin than in the case of the wild-type promoter fragment, for which Straney et al. (1989) reported t,hat CAP protein is still present on the open complex after 30 minutes incubation with heparin. The cause of this difference in stability is not certain. We postulate that the length of the substrate and the presence of bent DNA in close proximity to t.he CAP site may produce a high effective concentration of competitor DNA and contribute to the decrease in the stability of CAT’: recall that the rate of CAP dissociation from DKA depends on the square of the concentration of competitor DNA (Fried & Crothers, 1984). Tn agreement

with this idea, the speed at which (!AP is removed from the complex suggests that heparin may directly a,ffect the off-rate of the CAP protein in addition to acting as a competitor. Further study will be necessary to clarify this matter. (g) Electrophoretic

properties

of the closed

cmnpk~

Closed csomplexes are characterized by apprarante at low temperature and competitor-sensitivity (Hawley et al.. 1982; Stefano & (iralla, 1979; Straney & Crot.hers, 19%). At, 16°C’ for both formation and gel electrophoresis. we complex found one complex that was both compet,itorsensitive and CAP-dependent (Fig. 5(b)). This complex was not apparent when the reaction mixtures were incubated at a higher temperature. R4esults from other systems that parallel these findings include the promoter-specific complex reported at low temperature on bacteriophage T7 DNA (Williams & Chamberlin: 1977), and a corresponding complex identified on t.he wild-type tar: promoter (8trane.y et al., 1989). No such complex appears with

CAP-induced

CAP NTPs

I + t

3 t -

2 +

4 -

209

DNA Bending

417 Heoarin

4 O-O?

23 2.3 0.2

5 O-07

(mg/ml)

CAP

CAP

(bl

(al

Figure 7. (a) (‘AP-stimulation of open and initiated complexes. open and initiated complexes were formed on the construct that cont,ained the 12 bp linker as described in Experiment,al Procedures, and the reactions were run on a 4(V0 TBE gel at POT. Both the open and initiated complexes depend on the presence of CAP in the reaction mixture. Upon the addition of ?GTPs, the open complex is chased into the initiated complex (lanes 1 and 3). The initiat,ed complexes indicated were identified unambiguously by direct comparison with an autoradiogram of the gel and subsequent analysis of the transcripts contained in the complex (see Experimental Procedures). (b) The 2 open complexes differ with respect to thr presence or absence of CAP. Titration of heparin: open complexes were formed on 12 bp linker. supercoilrd D;CA at. 37’C. lmmediat~elg before loading on a gel heparin was added to a final concentration of: lane I. 4.66 mg/ml: lane 2. 2.33 mg/ml: lane 3. 02 mg/ml; lane 4. 007 mg/ml: lane 5. 0.07 mg/ml - (‘AI! upper open complex to shift into t,he lower open complex.

the csontrol plasmid

(no lac control

region)

under

t,hese conditions (Fig. 5(b), lane 8), or in the absence of C’AF’ (lane 7). Taken together, these results strongly indicate that this band reflects a closed complex originuting at the lac promoter. verSu8 linker length for A plot, of relative mobility

the closed complex is presented in Figure 3(a). Tn order to obtain complex bands that were sharp enough to yield reasonable measurements, closed complexes were run on 3.2% gels, as opposed to the 4 0,”/ gels used for the previous experiments. Under these condit,ions, the amplitude of the phasedependent variation of gel mobility is decreased due to the larger pore size of the gel matrix. An explanat,ion of the estimated correction may be found in Experimental Procedures. The variation in gel mobili+ of the closed complex is in phase with that of t.hr (-‘AT’ complex, which implies that the direction of thr brncl is unchanged.

(h) Relation between A-tract bend angle and the amplitude of the phase-dependent modulation of gpl mobility Given the significant variation in amplitude of t,he linker length-dependent mobility (Fig. 3), we felt it essential to address the question of whether the amplitude of gel mobility modulation continues to increase with increasing bend angle, or whether it might reach a maximum at a bend angle of 90”, as

High concentrations

of heparin cause the

predicted by the relation of mean-square end-to-end dist.anc:e to gel mobility. However. the theory of DX.4 mobility in gel electrophoresis is not sufficiently developed to draw conclusions about the magnitude of DNA bends based on gel mobility, especially for complex shapes such as those formed by these complexes. For these experiments, we constructed a. set of DSX molecules in which the kDNA from the present constructs was removed. and replaced with from three to nine A tracts repeated at 10.5 bp intervals, where each A tract consisted of six A. T base-pairs. The result was a set of DNA molecules with bends increasing from about 60’ t.o nearly 180”, shifted in phasing over one helical turn with respect to the CAP-induced DNA bend. (These bend angle values are based on our current estimate of the bend per A t.ract of IV, with an error of about 2” (Koo et nl.. 1990).) A plot of the modulation in gel mobility due t.o phasing of three, four and five A tract bends against) the CAP bend is shown in Figure 3(h). The magnitude of the gel mobility modulation reached a maximum for five A tracts. after which it was found to decrease slightly. The analysis is complicated by the fact that the shape of the curves generated by a plot of relative mobility versus linker length became irregular for molecules with six, seven and eight A tracts,

and became

regular

again

for nine A tracts,

as reported in detail by Zinkel (1989). It was found, however, that the amplitude of the phasemodulation curve was relatively insensitive to the

210

S. 5’. Zinkr.1 and Il. M. C’rother.u

(al CAP k7c promoter

t

-

+

t I

t 2

3

isomeric: sctt of’ D&A molec~ules. ‘I’hfb direct ion of’ t hri bend can 1beinf’errrd from thrl linker lengths that yield the minimum and maximum grl mohilitirs. The results reported here reflect a systematic* application of this strategy t,o intermediates in transcription initiation at the lac promot.rr. (b) IIN A is strongly

(b)

Open

-

Figure 8. Western blot of open complex. (a) Ethidiumstained gel of open complexes run at 37°C. Lane 11 complexes formed on DiVA containing a 12 bp linker + (‘Al’: lane 2. complexes formed on DKA containing a 12 bp linker - CAP: lane 3. complexes formed on control I)h’h (no lac promoter) + CAP. (b) Western blot of the gel shown in (a). The gel was elect,roblotted and probed with a monoclonal antibody to CAP. The presence of CAP is clearly visible in the open complex.

bend angle of the DXA molecule after the maximum swing was reached.

4. Discussion (a) Tracking

bend amplitude

and direction

The first gel electrophoretic approach to studying DNA bending was the circular permutation assay developed by Wu & Crothers (1984). When this assay was applied to the study of protein-DNA complexes such as CAP-RNA polymerase-DNA in which the dominant contribution to the gel mobility originates from frictional effects of the protein, the assay was found to be insensitive to changes in DNA structure (D. S. Straney & D. M. Crothers. unpublished results). We therefore developed a system in which a sequence-induced DNA bend was phased over one helical turn relative to a proteininduced bend in order to amplify small changes in the end-to-end distance of the DNA molecules upon which gel mobility depends (Zinkel & Crothers, 1987; Zinkel, 1989). In this assay, the extent of the DNA bend is a function of the amplitude of a plot of the relative gel mobility versus linker length for this

hunt in the oprn cwmpir.~

In an earlier model (Liu-Johnson et al., 1986), we proposed that t.he energy of DKA bending might be released in order to assist in formation of the open complex: a critical intermediate on the pathway to transcription activation: this model predict.s that DNA should no longer be strongly bent in the open complex. The objective of testing this model. and the intrinsic importance of the open complex, lead us t,o focus our primary attention here on the topology of IINA in that complex. Before addressing the interpretation of our experiments, we note that. Straney et al. (1989) found from DNase 1 digestion of the wild-type lae promoter bound to CAP and polymerase that the 5 bp alternation of protect.ion and enhancement of digestion sensitivity found in the CAP-DNA complex was retained in the ternary open complex. This pattern has been taken as evidence for DNA bending or curvature (Drew & Travers. 1985), and argues in favour of retention of the CAP-induced bend in the open complex. Two main results from the present experiments need to be taken into acc.ount in reaching a c-on&sion concerning DNA bending in the open complex containing (‘Al’: (I) the substantial increase in

electrophoretic

mobility

the

and

when CAP is removed from the near lack of phasein the elrctrophoretic mobility of sensit,ivity in phasing isomeric ])?;A molecules differing between their intrinsic: bend and the open complex: the weak but reproducible signal that rernains is shifted about 180” in phase from the pattern for CAP alone. This residual signal does not arise directly from intrinsic mobility variations from ant’ compltsx:

(2)

DNA isomer t,o another. hut rat,her results primarily from the analysis procedure. in which the mohilit~y of the complex is divided hy the mohilit,y of the

corresponding free I)SA in order t,o caorrecl for curvature in the DNA at sites other than thr IOCUS of protein binding (Zinkel & (‘rothers. 1987). In Table

1 we have collected

age reduction binding variety

dat,a on the prrcent

in gel mobility

of (‘AI’, of’ DNA

polymerase substrates.

tha,t result,s from

and tar repressor with and without

to a the

presence of bound polymerase. C!AP binding rvents are divided into two categories: (1) cases in which the CAP binding site is near the end of the DNA molecule. so that the effect of bending on gel electrophoresis is minimized (Wu $ Crothers, 1984); and (2) cases in which the binding site is nearer to the centre of t.he molecule, so that the electrophoretic effect, includes

the differential

consequence

of DNA

bending. The data in category (1) show that CAP bound near the end of a DNA fragment reduces the

CAP-induced

Table 1 I’errentage

decrease in ye1 mobility upon protein binding

DNA size (bp)

ix3 (I.\‘5 open’) -430 (open-CAP) E. (‘Al’

Mobility decrease P”Y

Gel conditionb

61 68

4Y,. 41 : I. TUE -I’&. 34 : 1, TUE

binding to polymmw

- 430 (initiated) - +:10 (open)

26 “4

Reference’

4 2

cmplex 44,. 39 : 1, TEE 4qb. 39 : 1, THE

2 1

AThe mobility dec~rease is defined as the difference between mobilities before and after protein binding, divided by the mobility before binding. and multiplied by 100 to yield a percentage value. h’l’ht* gel conditions are given as percentage arrylamide, ratio of acrylamidr to his-acrylamide, and buffer. TUE buffer is d&net1 in Experimental Procedures and TE buffer is 10 mnr-Tris. 1 mwEDT.4 (pH 7.4). ’ References: 1. Zinkrl & (‘rothers (1990); 1, this work; 3. Fried C (‘rothers (1981): 1. Straney & (‘rothers (1985). “\Vhen the I)?iL% size is denoted as approximate (-). the results have hrru averaged over 1 full cycle of a set of phasing isomers. ‘The uumherp Iudicaatrx the number of repressor trtramers Ixnlnd. ‘This value refers to the upper open complex.

electrophoretic mobility by 9% to 14%, varying only marginally with the length of A-tract, composition of the DNA. In contrast, when binding is near the DNA centre, category (2), the mobility is reduced by about 36%. which is again relatively independent of DNA size or A-tract composition. Xote that these percentages depend on gel concentration and composition, which must be held constant for valid comparisons. Tn considering the differential mobility effect due to CAP in polymerase--CAP-DNA complexes, it should be recognized that CAP binding occurs not to free DXA but to an existing protein-DNA complex. The caomparat,ive data for binding successive lac repressor tet.ramers to operator-containing DIVA (Table 1C) demonstrate that the percentage retardation diminishes as more and more protein is bound. Hence. it is expected that the mobility retardation due to simple binding effects when CAP is bound to a polymerase-DNA complex should be

DNA Be,nding considerably smaller than the 9 to 147, range found for binding to naked DNA when the influence of bending is removed. Note t’hat the total retardation due to binding three Zac repressors (Table 1C) is comparable to the retardation due to binding a single polymerase (Table 1D). Table 1E lists the mobility reductions observed when (1AP binds to initiated (26?/,) and open (2404) complexes. The effects are quite similar, and much larger than expected for simple CAP binding under these conditions. Hence, these results, along with the earlier footprinting data on both complexes (Straney et al., 1989), strongly support the view that CAP is responsible for substantial DNA bending in both open and initiated complexes. Since we find that the CAP bend is maintained and even increased in formation of an open complex, we conclude that the bend energy is not expended to help melt the promoter DSJ’A. as postulated earlier (Liu-Johnson et al.. 1986). A tighter bend in the open complex would be energetically cost,ly. but could be readily compensated for by an and/or strength of increase in the number (‘AFpolymerase contacts. The increased stability of CAP to competition by heparin in the open complex (Straney et al., 1989) suggests the presence of strong protein-DKA eont.acts, as well as possible (‘AI’-polymerase contacts, making this complex a very stable int’ermediate. Increase of t,he DNA bend angle beyond that due to CAP alone could be achieved structurally in several ways, for example by tightening the contact bet,wet:n DNA and CAP or by dist.orting DKA in the region rontactrd by polymerase. Recent experiments on digestion of Zac open caomplexes with mic~roc~occal nuclease (Zhang & (iralla. 1989) revealed regions of hyper-reactivity at both the upstream and downstream boundaries of the complex. suggesting that the I)XA at, t.hr boundaries of this complex has been distorted such that the backbone is exposed, facilitating micrococcal nuclease attack. The surrounding sequences are not particularly disposed to adopt a bent configuration based on the dinucleotide parameters reported by (iartenberg 6t (‘rothers (1988). In view of the a.bove evidence for bending in the open cbomplex, one model to explain this phenomenon is that a kink forms in order to relieve a locaal torsional stress due t 0 unwinding. (t,) I nterpretntion

of the phase-dependrnce

of

the gel mobility The remaining feature of our results. a very small phase-dependence of the mobility of the set of isomeric open complexes containing CAP, might be mistaken as evidence for lack of bending in the complex if t,he result were taken out of context. However. there are two kinds of molecules in which the variation of end-to-end distance is effectively zero over the set of phasing isomers: those in which the bend angle is O”, and those in which it is 180”. Other results that support substantial bending in

the open complex cont.aining (IAl’ dic*t,ate choicte ot the latter possibility. This option also offers a simple explanation for the 180” phase shift of’ the stnitll mobility modulation t,hat remains. namely that thr bend angle is increased t’o a value slightly greater than 180”. (Sate that when 1 of t’he 2 bend angles itI a phased pair is increased ak~ovc 180”. simpltl geometric considerations indicat’e that the rriti-toend distance. on which mobility depends (I,umpkin & Zimm. 1982: .J. Ifrak & I). Xi. (‘rothers, unpub lished results). (*an hecome shorter in the tmrr,.s t ha11 in the cis isomer.) %Tr tnade an e@ort to mimic this eff& with large A-t,ract bends hy increasing the numhrr of’ A tracts in the bend that was placed in variable phasing with t,hr (‘AT’ bend. As Figure 3(h) shows: the amplitutlri of’ t.he phase modulation curve increased when t hri number of A t,racts is increased from three t)o five: the latter value should produce a bend of roughI> 90”. Ikyontl this point. the curves diminished in amplitude but hecame irregular in phaxillg as rrported in detail by Zinkel (1989); the at~om~1ou~ phase effect may he related to thrb ability of the gel to separate DSA diasteromers, as documented 1)~ ,I. I)rak & I). M. Crothers (unpublished results) for simpler constructs containing only .A-tract bends. \Vit.h as many as nine A tracts phased against the (“Al’ bend, we found that the amplitude of the phase-modulation curve diminished, but did not zero, as was the case when the large approach (:Al’+ polymerase-induced bend was phased against the BDNA bend (Fig. 3(e)). We suspect that the mobilities of DNA molecules coni parative cont,aining very large bends are greatly affected by t,he difference between relatively flexible A-tract, bends and the rigid bend around CAP-polvmerase, making A-tract bends poor models for proteininduced bends in such circumstances. I)NA unwinding could also cause a shift in the phase modulation plots of Figure 3. but only if the unwinding occurs between the bends. The phase shift c.annot be at)tributed to unwinding in the open c*omplex, since the region of the promoter t,hat is unwound in that complex does not lie between the (‘Al bend and the kI)I\;A bend.

(d) Hending

in the closed

complex

Our experiments (Fig. 3(a)) show that, the phase modulat.ion of mobility in t,he CAP-induced closed complexes is reduced in amplitude but unaffected in phase. relative to t,he properties of the complex with (“AP alone. Since in this case we have neither foot.printing data nor knowledge of the mobility differencae between closed complexes with and without (:AP, it is dificult to assess the significance of the phase modulation amplitude. However: we find it. plausible to propose that the lower amplitude results from a progressive tightening of the bend as transcription initiation proceeds from the CAP complex through closed complex, and finally to 0lJen complex.

LTnderstanding

the mechanism

of gene a,c*tiv:tt~iotl by the long-standing observation t,hat its l)NA binding sites t~~cl trot have a common location relative to IJol~rnt~rasc~ recognition or start signals (de(‘rombrugge p’f crl.. 1984). In particular, in thr two major motifs r~~pr(~sentad try thr lr~c and go,1 promoters. t.hta (‘Al’ c*csntrfb is pcjsitioned 61 bp and 41 hp. respectiveIT. from the mRSA start site. Occupancy by (IAP of a fixed binding sitcx on ljolymrrasr would require s(kvt?rc I)NA tlist.ort~ion in at lftasl one of’ I Itcw owes. assuming the spat.ial relation between ( ‘A I’ and the polymerasc active site is thr same in t.h(l /UC and q”/ promoters. Other data also support. the idea of IjN-4 bending in the polymerase complex. For example. the lowresolution structure of RXA polymt~rase reported by Darst rt nl. (1989). tJy analogy with t hr structure indicates a of I)NVA ljolymrrase. ljutative - fall naturally at the -41 and -61 loci when IJNA is curved around polymerase. It should IJ~ rmphasizcd that simult,aneous occupancy of both sitrs w~nld require substantial distort,iotr of tht, (‘AI’mI)NA c*omplex, and would tltjt. necessarily IJtA fBVOUri+.lJl?, nor does t.he model require tloutjlo o~~~uljat~~~~~. Rather. it seems more likely that, polymrrasr itself enforces the curved I)SA path tha,t is followr~tl f’rom

Kay(:AP has I~oen complicat’ed

CAP-induced

DNA

Bending

213

CAP

,: Figure 9. A structural model of the polymerase-CAPDXA complex. Two potential CAP binding sites are shown in their relative placement in the lac and gal promoters. Generally, only one of these sites would be occupied, but the DKA might follow a trajectory as though curved around CAP in both sites The resulting DKA topology is analogous to that found at the terminal loop of an interwound negative superhelical domain, although bending in the CAP-polymerase complex is tighter than would be expected in uncomplexed superhelical DNA. Formation of this special structure may facilitate transduction of superhelical stress to unwinding of the promoter during conversion from closed to open complexes.

Open

Closed

/

htmted

CAP at -61 in the lac promoter to the polymerase active site. This model accounts naturally for our results, which require a DNA bend in the open complex that is about twice as large as that, induced by CAP alone, estimated t.o be about 100” (Zinkel & Crothers, 1990). (f) IjLVd

bending

in transcription

initiation

Figure 10 summarizes our model of the series of changes in D9A bending that occur during transcription initiation. The CAP-DNA complex, shown in this instance with the additional A-tract bend, binds polymerase first in the closed and then in the open complex, with progressive tightening of the bend during the process. Finally, in formation of the initiated complex, polymerase escapes from the promoter, and the DNA bend reverts to the normal CAP-induced form: if heparin is added, CAP binding and the associated bend are lost. At this time we are able to identify three possible (non-exclusive) roles for DXA bending in act,ivation of transcription. which we consider here briefly. (1) It seems likely that DNA curvature is essential for establishing the contacts between CAP and polymerase that, are required for enhanced polymerase binding. The existence of such contacts is implied by stabilization of a cGMP-liganded CAP* (cAMPindependent mutant)-DNA complex in the presence of RKA polymerase (Ren et al., 1988). Additional evidence for such contacts has been report’rd by Straney et al. (1989): who showed polymerase-mediated stabilization of CAP in the open complex formed at the wild-type Zac promoter. According to the model. the result of these contacts is to cause the overall DNA bend to tighten. decreasing the amplitude of t,he phase-dependent modulation in gel mobility.

Figure 10. Proposed model for the change in DNA bending during transcription initiation. CAP protein is represented as a dimer of 2 circles. DKA polymerase is represented as an oval. and DNA is represented as a line. The constant

section

of the DSA

molecule

(kDKA)

drawn after the CAP complexes. The CAP-induced bend is maintained through the formation an open complex. but is released upon stably initiating complex.

is not

Dl\A

of a closed and formation of a

(2) \$l’e propose that the topology of DSA in the CAT’-polymerase-DNA structural model (Fig. 9) results in placement of the promoter at a terminal loop in an interwound negative superhelical domain. Because DNA bending is maximal in such a loop. provision of a bending site at the promoter would tend to localize the promoter to this specific site. Interaction of DNA upstream from the CAP site with the back side of polymerase during the transition from closed to open complex may facilitate the unwinding process, perhaps by coupling to the energy available from negative superhelital tension. Such a mechanism of activation should not, and does not apply to the Zac promoter on linear 1~54 fragments, since a removal of DSL4 upstream of the CAP site does not affect activation (Straney ef al.. 1989). The proposed t’opology is consistent, with the ability nf a sequence-directed bend substituting for the (“Al’ site to stimulate transcription from the gal promoter in rho (Bracco et al.. 1989) and from the lac promot)er on superhelical templates in vitro

214

((iartenberg & (‘rothers, 1991 ). Furthrrmorr. the studies of Meikeljohn & Gralla (1989) indica,te that (YAP and supercoiling are able to activate transcription in a synergistic manner at, the superh&cities found in viva. (3) 1Z’r propose that I)XA bending fa,cilitates polymerase escape from the promoter. In view of the high stability of the CAP-polymerase-promoter complex. it is perhaps surprising that the reaction is not rate-limited at the point of movement int,o the initiated complex. Strong bending in the open complex compared to the initiated complex suggests a plausible role for the energy st’ored in the IIS.4 hc,nd: the tendency of the promoter DSA to straighten provides a force that, 11ulls both l)?;A and (AI’ in a direction opposite to that in which the polymerase is attempting to move. The result ivill be to destabilize (‘AP-DNA and (!AP--polymrrasr contacts. disrupting the open complex and allowing polgmerase to move down the DNA. (Our results do not allow us to distinguish the 2 possible sites of (*ontact breakage. either between (‘AT’ and DSA. or between (‘AI’ and polymerase; experiments reported by Straney rt al. (1989) provide support for movement of (‘AT’ with polymerase into thr init,iated complex.) A role for the (‘Al’ bend in facilitating movement of RSA polymerase into an initiated complex is supported by kinetic studies of the maZT promoter (Menendez rt crcl.. 1987). which has been shown to be more sensitive than either Inc or q~,l, promot,ers to the superhelical state of DNA in ~~iw (Sanzey. 19X7). This work was supported by grant GM34205 from the Xational Institutes of Health. We gratefully acknowledge the following for the gift of reagents: Dr Joseph Krakow for the gift of anti-CAP monoclonal antibody mAb 64B4; Dr David Straney and Dr Susan Straney for purified RXA polymerase holoenzyme; Dr Abraham Brown for Iturified CAP protein. We thank Dr Henri But for communicating results in advance of publication.

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