The X repressor contains two domains

Transcription

1 Proc. Ntl. Acd. Sci. USA Vol. 76, No. 4, pp , April 1979 Biochemistry The X repressor contins two domins (scnning clorimetry/ppin digestion/oligomeriztion/dna binding) CARL 0. PABO*, ROBERT T. SAUER*, JULIAN M. STURTEVANTt, AND MARK PTASHNE* *The Biologicl Lbortories, Hrvrd University, Cmbridge, Msschusetts 02138; nd tdeprtment of Chemistry, Yle University, New Hven, Connecticut Contributed by Julin M. Sturtevnt, Jnury 9,1979 ABSTRACT Ppin digestion of the A phge repressor produces two frgments tht re reltively resistnt to further digestion. One includes the mino terminus (residues 1-92) nd the other the crboxyl terminus (residues 132-). Clorimetry shows tht the mino-terminl frgment dentures ner 50'C nd tht the crboxyl-terminl frgment dentures ner 70'C. Intct repressor undergoes two denturtions, one ner 500C nd nother ner 70'C. These nd other dt show tht X repressor consists of two domins joined by "connector" 40 mino cids long tht is sensitive to proteses. The mino-terminl domin binds DNA, nd the crboxyl-terminl domin oligomerizes. The bcteriophge X repressor is protein (monomer Mr 26,000) tht medites both positive nd negtive control of gene trnscription. Repressor monomers re in equilibrium with dimers nd higher oligomers. Repressor dimers bind strongly to specific DNA sequences clled the X opertors, thereby controlling gene expression (for review, see ref. 1). The repressor is inctivted by proteolytic clevge in ivo (2) if Escherichi coli cells contining repressor re treted with ultrviolet light or, pprently, with ny ctivted crcinogen (3). This clevge rection hs been observed in vitro (4, 5). The mino cid sequence of the X repressor hs been determined (6), s hs the nucleotide sequence of its gene (ci) (7). We show in this pper tht ech repressor monomer contins two domins of similr size, one from the crboxyl nd the other from the mino terminus, joined by "connector" region of 40 mino cids. The domins hve distinct functions: the mino-terminl domin binds to DNA nd the crboxyl-terminl domin oligomerizes. The "connector" is sensitive to proteolysis in vitro nd lso contins the site sensitive to the physiologiclly importnt proteolytic ttck mentioned bove (M. Ross, R. T. Suer, nd M. Ptshne, unpublished dt). MATERIALS AND METHODS Enzyme Digestions. X Repressor (pproximtely 1 mg/ml), purified s described (8), ws cleved with ctivted ppin (E:S, 1:200, wt/wt) (9) (Worthington) t 220C in buffer B nd the rection ws terminted t vrious times with Koshlnd's regent III (10). Preprtive digestions were crried out for 30 min. Repressor Frgments. Frgment d ws seprted from frgments -c by chromtogrphy on SP-Sephdex (C-25). In buffer A, frgments, b, nd c flowed through the column. Frgment d ws eluted with buffer A plus 100 mm KC1, nd ws purified further by elution from hydroxylptite column with liner grdient of M buffer C nd, finlly, by gel filtrtion on Sephdex G-75 (superfine) in buffer A plus 200 mm KCl. Frgments nd b were purified from the mixture of frgments, b, nd c by filtrtion on Ultrogel AcA 44 in The publiction costs of this rticle were defryed in prt by pge chrge pyment. This rticle must therefore be hereby mrked "dvertisement" in ccordnce with 18 U. S. C solely to indicte this fct. buffer A plus 200 mm KC1. Frgment c eluted contminted with frgment, nd the contminting frgment ws subsequently converted to c by further ppin digestion. Finl purifiction of c ws chieved by rechromtogrphy on AcA 44. Purified frgments were chrcterized by utomted Edmn degrdtion nd mino cid nlysis s described (6). Gel electrophoresis in 13.5% polycrylmide gels ws s described by Lemmli (11). Sedimenttion equilibrium experiments were performed by using model E Spinco nlyticl ultrcentrifuge nd the stndrd short column procedures (12). Repressor frgments were dissolved t 56,.M in buffer D. The smples were centrifuged in n An-D rotor t 11,890 rpm t 220C for frgments, b, nd c nd t 24,088 rpm t 20 C for frgment d. Equilibrium ws estblished within 24 hr. Frgment concentrtions in the cell were determined by opticl scnning t 280 nm. Prtil specific volumes were clculted s described (13). Filter binding ws performed t 0 C in buffer E s described (8). Plsmid PORI is derivtive of pmb9 tht crries the X opertor site OR1 (A. Johnson, personl communiction). Plsmid pop203-1 is isogenic with POR1 except tht it lcks the X opertor (F. Fuller, personl communiction). These DNAs were lbeled by nick trnsltion s described (14). Plsmid DNA ws used t 1 nm. Clorimetry. Denturtion of repressor nd repressor frgments ws observed in differentil scnning microclorimeter t protein concentrtions of 1-3 mg/ml in buffer F. Enthlpies for individul trnsitions were clculted s described (15, 16) Ḃuffers. Buffer A: 10 mm Tris-HCI, ph 8.0/2 mm CCl2/0.1 mm EDTA/5% glycerol. Buffer B: buffer A plus 0.1 mm dithiothreitol, 2 mm cysteine, 200 mm KCI, nd 0.4 mm EDTA. Buffer C: 1 M buffer C contins 0.5 M KH2PO4 nd 0.5 M K2HPO4. Buffer D: 20 mm Tris-HCI, ph 8.0/200 mm KC1. Buffer E: 50 mm N ccodylte-hcl, ph 6.9/2 mm CCI2/0.1 mm EDTA/5% dimethyl sulfoxide/100,ug of bovine serum lbumin per ml/10,g of chicken blood DNA per ml. Buffer F: 50 mm N3 citrte titrted with citric cid to ph 5.5. RESULTS Proteolytic Dissection of X Repressor. Prtil digestion of X repressor with ppin gives four mjor frgments tht cn be detected by gel electrophoresis. Fig. 1 shows time course of ppin digestion of purified X repressor. The frgments lbeled nd d pper first. By 22 hr, only frgments c nd d remin in substntil mounts. Ech of the four frgments ws purified (Fig. 2), nd the composition nd mino-terminl sequence of ech were determined. These dt (Tbles 1 nd 2) nd the known sequence of repressor (6) determine the origin of ech frgment (Fig. 3). Frgments (residues 93-), b (residues 122-), nd c (residues 132-) re crboxyl- Abbrevition: ts, temperture sensitive. 1608

2 Biochemistry: Pbo et l. Proc. Ntl. Acd. Sci. USA 76 (1979) 1609 p pp - C. mw~~ 4m-Ad W m "M d -- - I fti _w WV w qw F-- _ Time, hr FIG. 1. Time course of ppin digestion of repressor. The digestion ws stopped t the vrious times indicted nd the products were nlyzed by sodium dodecyl sulfte gel electrophoresis. R, position of intct repressor;, b, c, nd d, mjor frgments. The dye is bromphenol blue. NH2 r 132 c -_ COOH ~~d NH2 NH 92 FIG. 3. X Repressor frgments nd ppin cutting sites. Upper line represents the mino cid residues of intct X repressor. Shorter lines below indicte the extent of ech frgment, nd the rrows show the principl sites cut by ppin. All numbers inthe figure refer to residue numbers of intct repressor. thlpy of 140 kcl molh'. The enthlpies for the two trnsitions re so lrge tht they must be due to unfolding of the repressor rther thn to chnges in its oligomeric stte (ref. 18 nd refs. cited therein). The mino-terminl frgment (d) undergoes single trnsition ner 51'C (Fig. 4B) with n enthlpy of 80 kcl molh'. Ech of the three crboxyl-terminl frgments undergoes single trnsition in the rnge of C, nd in ech cse the unfolding bsorbs kcl molh'. The denturtion trnsition of the mino-terminl frgment (d) resembles the low-temperture trnsition of repressor, wheres the trnsitions of the crboxyl-terminl frgments (, b, nd c) resemble the other repressor trnsition. We conclude tht the trnsitions tht repressor undergoes ner 500 nd 70'C correspond, respectively, to the unfolding of the mino-terminl A T CL 1 cl K-'g' i i b -' COOH -_ COOH -' COOH terminl peptides, wheres frgment d (residues 1-92) is n mino-terminl peptide. Our ssignments re consistent with the reltive mobilities of the frgments on sodium dodecyl sulfte gels nd with the order of ppernce of the frgments during ppin digestion (see Fig. 1). Extensive digestion of repressor with elstse, thermolysin, or subtilisin produces two mjor frgments with electrophoretic mobilities similr to those of frgments c nd d, wheres trypsin extensively degrdes the molecule (not shown). Ppin clevge of repressor thus yields stble frgment (d) from the mino terminus nd nother (c) from the crboxyl terminus. The sequence of ech of these frgments contins mny potentil ppin clevge sites (17), nd their resistnce to ppin clevge indictes tht they re stbly folded. The experiments reported below provide dditionl evidence tht these frgments re folded nd show tht the corresponding regions of whole repressor re domins tht denture independently s repressor is heted. Therml Denturtion of Repressor nd Its Frgments. Clorimetry ws used to study the therml denturtion of X repressor nd of the four frgments produced by ppin digestion. Repressor undergoes two mjor endothermic trnsitions (Fig. 4A). The first occurs ner 490C nd hs n enthlpy of 63 kcl mold, while the second occurs ner 730C nd hs n enbm d > _ Dye FIG Purified repressor frgments. Four microgrms of purified frgment d (lne 4) nd 2,ug ech of purified frgments, b, nd c (lnes 1, 2, nd 3, respectively) were nlyzed by sodium dodecyl sulfte gel electrophoresis Temperture, 'C FIG. 4. Denturtion of repressor (A) nd repressor frgments (B) in scnning clorimeter. Ech frgment ws studied over the entire temperture rnge, nd only one trnsition ws observed for ech. The clculted enthlpies (in kcl mol-1, 1 cl = joules) for the trnsitions re: repressor (trnsition ner 490C), 63; repressor (trnsition ner 73CC), 140; frgments, b, c, nd d, 115, 130, 125, nd 80, respectively.

3 1610 Biochemistry: Pbo et l. Proc. Nti. Acd. Sci. USA 76 (1979) Tble 1. Frgment b c d Amino-terminl sequences of repressor frgments Amino-terminl sequence Met-Gln-Pro-Ser-Leu-Arg-Ser... Thr-Lys-Gly-Asp-Al-Glu-Arg-Trp-Vl... Thr-Thr-Lys-Lys-Al-Ser-Asp-Ser-Al... Ser-Thr-Lys-Lys-Lys-Pro-Leu... Frgment d is homogeneous, but ech of the frgments, b, nd c contined minor sequences (20-30%) resulting from clevge of one to three residues from the mino-terminl ends of the peptides shown. nd crboxyl-terminl domins. These domins re sufficiently independent to denture seprtely in the intct molecule nd to retin their therml stbility when seprted by proteolysis. It my be noted in pssing tht some of the denturtion curves in Fig. 4, prticulrly tht for frgment, re considerbly shrper thn usully observed for the therml unfolding of proteins in queous solution. The full significnce of this is not understood. DNA Binding of Frgments. Of the four frgments of repressor, only the mino-terminl frgment (d) binds DNA, s mesured by its bility to retin 32P-lbeled DNA to nitrocellulose filters (Fig. 5). This DNA filter binding is not specific: DNA with or without X opertor is bound eqully well. Micromolr concentrtions of frgment d re required for hlfmximl retention of the DNA. In contrst, under similr conditions, intct X repressor t 7 X M retins hlf of the opertor-contining DNA but virtully none of the control DNA on the filter (not shown). We show elsewhere, using more sensitive ssy (19), tht frgment d binds specificlly to the X opertors. This specific binding occurs t concentrtions of the frgment below those required to bind DNA to the filter. Evidently this frgment d-opertor complex is not detected efficiently in filterbinding ssy. It is possible tht the hlf-life of the complex is too short for efficient retention on filter or tht frgment d remins bound to the opertor but tht binding t the opertor site lone is not sufficient to trp the complex on filter. We ssume tht the nonspecific filter binding of DNA by frgment d results from simultneous binding of the frgment to mny Tble 2. 'S0 z 0.5X 10-' 1.0 X lo" Frgment d, M FIG. 5. Nitrocellulose filter binding of DNA. 32P-Lbeled plsmid DNAs with (0) or without (A) X opertor were mixed with frgment d nd pssed through nitrocellulose filters. A single curve fits both sets of points, nd the plteu of this curve corresponds to retention of 80% of the DNA. At similr concentrtions, frgments, b, nd c did not bind plsmid DNA to the filter. sites on the DNA. Similr results of DNA-binding experiments hve been reported with n mino-terminl frgment isolted from the lc repressor. Filter-binding experiments revel only nonspecific binding (20), wheres more sensitive methods show tht this frgment binds to the lc opertor specificlly (21). Oligomeriztion of Frgments. Sedimenttion equilibrium of repressor frgments t high concentrtion (-s50,um) revels tht two of the three crboxyl-terminl frgments form oligomers nd tht the mino-terminl frgment does not (Tble 3). At these concentrtions, intct repressor hs n verge Mr corresponding to n oligomeric species between tetrmer nd n octmer (22). Tble 3 shows tht most of the frgment c is dimer, frgment is higher oligomer, nd frgments b nd d re monomers. We hve not exmined the stte of ggregtion of these frgments t physiologicl concentrtions (_10-7 M), where intct repressor is primrily dimeric (23, 24). In experiments not shown, we hve found tht frgments nd c elute from sizing columns in the positions expected for dimers or higher oligomers nd tht frgments b nd d elute s expected for compct monomers. We hve lso found tht ddition of frgment c to solution contining repressor reduces Amino cid compositions of repressor frgments IL (93-) b (122-) c (132-) d (1-92) Theory Found Theory Found Theory Found Theory Found Asx Thr Ser* Glx Pro Gly Al Cys 3 ND 3 ND 3 ND 0 ND Vl Met Ile Leu Tyr Phe His Lys Arg Trp 3 ND 3 ND 2 ND The crboxyl-terminl sequence of frgment d ws confirmed by crboxypeptidse degrdtion (not shown). ND, not determined. * Serine vlues were corrected by 15% to ccount for destruction during hydrolysis. A t o e0 I,0-II %r d%-5 0 td-

4 Biochemistry: Pbo et l. Tble 3. Sedimenttion equilibrium nlysis f repressor frgments Proc. Nt!. Acd. Sci. USA 76 (1979) 1611 Frgment (residues) Mr clculted from sequence Prtil specific vol V, cm3/g Averge solution Mr (Solution Mr) * (monomer Mr) (92-) 15, ,300-77, b (122-) 12, , c (132-) 11, , d (1-92) 10, , For frgments b, c, nd d, the plots of In c ginst r2 were stright lines, indicting the presepce of single predominnt oligomeric species. For frgment, this plot showed significnt curvture, indicting the presence of severl oligqmeric species. the bility of repressor to bind opertor DNA to filter. We ssume tht repressor inctivtion is cused by the formtion of hybrid dimers of frgment c nd intct repressor. Hybrid dimers would contin only one DNA binding site nd would not specificlly bind opertor DNA to filter. An nlysis of the effectiveness with which frgment c inhibits repressor binding suggests tht hybrid dimer is lmost s stble s repressor dimer. Thus, frgment c my contin the sme dimeriztion site s does intct repressor. We do not know why frgment b, which is slightly lrger thn frgment c, fils to oligomerize. [It is possible tht the mino-terminl 10 residues of frgment b (residues , not contined in frgment c) re not folded s they would be in ntive repressor nd tht these 10 residues prevent the formtion of frgment b dimers. ] DISCUSSION The experiments reported in this pper suggest tht the X repressor contins two domins joined by protese-sensitive "connector" region of 40 mino cids. One domin oligomerizes nd the other recognizes DNA. The rgument is s follows. When X repressor is heted, it dentures in two steps t tempertures seprted by over 20'C, s observed in scnning clorimeter. Proteolytic clevge of repressor produces two reltively stble frgments, ech of whose denturtion profile in the clorimeter mimics one or the other of the trnsitions observed with intct repressor. One of these frgments includes the mino terminus (residues 1-92) nd the other includes the crboxyl terminus (residues 132-). The former binds DNA but does not oligomerize even t concentrtions (50,uM) much higher thn tht t which hlf the repressor exists s dimers (KD - 2 X 10-8 M) (23,24). The ltter oligomerizes but does not bind DNA. Experiments reported elsewhere (19) show tht the mino-terminl domin mkes specific contcts with X opertor DNA nd tht this interction medites positive nd negtive control of gene function. The crboxyl-terminl domins oligomerize. We believe this stbilizes the repressor-dna complex by linking the energy of binding of monomers, both of which, we imgine, contct DNA in the functionl dimer. The lc repressor, stble tetrmer, contins n mino-terminl region tht recognizes DNA nd crboxyl-terminl region tht oligomerizes (20, 21, 25). The "connector" region between the two domins of repressor contins t lest three sites sensitive to ppin nd lso contins the site cleved by protese in E. coli. Apprently the reca gene encodes n ATP-dependent protese tht is stimulted by het in certin bcteril mutnts or by tretment of wild-type E. coli with ctivted crcinogens (2-4). In prtilly purified cell extrcts n ATP-dependent protese, presumbly the reca protein, cleves X repressor between residues 111 nd 112 (M. Ross, R. T. Suer, nd M. Ptshne, unpublished dt). This clevge inctivtes repressor becuse the (reltively low) concentrtion of mino-terminl frgment ordinrily found in crcinogen-treted cells is not sufficient to mintin repression. The response of E. coli to ctivted crcinogens, the so-clled SOS response, includes vriety of chnges in gene expression, of which X induction is but one exmple (26). Presumbly the reca protein cleves other repressors tht control genes ctivted during SOS. It remins to be seen which conrrmon structurl fetures of these vrious repressors render them subject to clevge. Our results confirm n erlier surmise bout X repressor structure bsed on genetic experiments. Lieb (27) nd Nono nd Gros (28) found tht repressors rendered temperture sensitive (ts) by muttion in the mino-terminl portion regined ctivity in vivo when the cells were returned to lower temperture. In contrst, repressors bering ts muttions in the crboxyl-terminl portion did not regin ctivity. In ddition, severl ts repressors ltered in the crboxyl-terminl region complemented severl ts repressors ltered in the mino-terminl region, but ts repressors bering muttions in the sme region of the protein usully did not complement (29). Moreover, Oppenheim nd Noff (30) found tht trnsdominnt cimutnts re locted only in the mino-terminl third of ci. These mutnts, by nlogy with lc i-d mutnts (25), re pprently deficient in DNA binding but re functionl in oligomeriztion. Lieb (29) suggested, therefore, tht the crboxyl-terminl section of repressor promotes subunit ggregtion nd tht the mino-terminl prt binds to the opertor. Both the isolted mino- nd crboxyl-terminl domins (frgments c nd d) hve been crystllized in forms menble to nlysis by x-ry crystllogrphy (unpublished dt). We expect tht the solution of these structures, prticulrly tht of frgment d, will illuminte the mechnism by which the X repressor binds tightly to its opertors. We thnk Leroy Liu, Kshyr Jvherin, nd Jmes Wng for their help in the sedimenttion equilibrium experiments, nd Guido Guidotti nd Alexnder Johnson for helpful discussions. This work ws supported by grnts from the Ntionl Institutes of Helth nd the Ntionl Science Foundtion. 1. Ptshne, M., Bckmn, K., Humyun, M. Z., Jeffrey, A., Murer, R., Meyer, B. & Suer, R. T. (1976) Science 194, Roberts, J. W. & Roberts, C. W. (1975) Proc. Nt!. Acd. Sci. USA 72, Moreu, P., Bilone, A. & Devoret, R. (1976) Proc. Ntl. Acd. Sci. 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