DNA-Looping and Enhancer Activity: Association Between

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Proc. Nati. Acad. Sci. USA Vol. 87, pp. 5504-5508, July 1990 Biochemistry DNA-looping and enhancer activity: Association between DNA-bound NtrC activator and RNA polymerase at the bacterial ginA promoter (positive regulation/protein-protein interaction/transcription regulation) WEN SU*, SUSAN PORTER*, SYDNEY KUSTU*t, AND HARRISON ECHOLS* *Department of Molecular and Cell Biology and tDepartment of Plant Biology; University of California, Berkeley, CA 94720 Communicated by I. Robert Lehman, May 7, 1990

ABSTRACT The NtrC protein activates transcription of promoter site (or with other proteins that contact polymer- the ginA operon of enteric bacteria by stimulating the forma- ase) (DNA-looping model) (11-13, 16). tion of stable "open" complexes by RNA polymerase (oM- The DNA-looping model for enhancer action has been holoenzyme form). To regulate the ginA promoter, NtrC binds considered attractive for several reasons. The interaction to sites that have the properties of transcriptional enhancers: between DNA-bound proteins has been for some time an the sites will function far from the promoter and in an established principle for control of site-specific recombina- orientation-independent fashion. To investigate the mechanism tion and DNA replication (12, 16-19). For transcriptional of enhancer function, we have used electron microscopy to control, DNA-looping was proposed initially as an appropri- visualize the interactions ofpurified NtrC and RNA polymerase ate way to visualize the interaction oftranscription regulators with their DNA binding sites and with each other. Under bound at separated DNA sites (6, 13, 20-23). More recently, conditions that allow the formation of open complexes, about the DNA-looping model for transcriptional regulation has 30% of DNA molecules carry both RNA polymerase and NtrC been supported by several more direct types of experiments. bound to their specific sites. Of these, about 15% form looped DNA-looping interactions between homologous regulatory structures in which NtrC and the RNA polymerase-promoter proteins have been visualized by electron microscopy (24- complex are in contact. The length of the looped DNA is that 27). Recent biological and biochemical experiments have predicted from the spacing that was engineered between the used mutant repressors to correlate these looping interac- enhancer and the ginA promoter (390 base pairs). As expected tions directly with negative regulation in vivo in the gal and for activation intermediates, the looped structures disappear lac operons (28, 29). For enhancer systems, recent work has when RNA polymerase is allowed to transcribe the DNA. We shown that an enhancer site can stimulate transcription in conclude that the NtrC enhancer functions by means of a direct trans to a promoter if the two sites are closely associated by association between DNA-bound NtrC and RNA polymerase DNA catenation or protein-mediated tethering (10, 30, 31). In (DNA-looping model). Association of DNA-bound proteins the work reported here, we have demonstrated the central appears to be the major mechanism by which different types of biochemical prediction of the DNA-looping model for posi- site-specific DNA transactions are localized and controlled. tive regulation from an enhancer: the heterologous interaction of the DNA-bound regulatory protein with RNA poly- Enhancer sequences have been defined by their function in merase at the promoter. We report an interaction between activating transcription from relatively long distances in an enhancer-bound NtrC protein and RNA polymerase that is orientation-independent manner (1, 2). There are many well- mediated by a DNA loop. characterized examples of enhancers controlling eukaryotic cellular and viral genes (3-5). Although initially defined in eukaryotes, regulation of transcription from distant sites has MATERIALS AND METHODS been observed for many prokaryotic promoters (6, 7). The DNA. Two DNA restriction fragments were used. The first, site for positive regulation of the glnA operon of enteric which carried the wild-type ginA fragment [453 base pairs (bp); bacteria is a particularly well-defined example of a prokary- Fig. 1 Upper], was a HindIII-BstNl fragment from the plas- otic enhancer (8-10). To investigate the mechanism of en- mid pJES125 (P. Wong and S.K., unpublished work). The hancer action, we have studied the activation oftranscription second fragment (782 bp, Fig. 1 Lower) carried a 347-bp from the glnA promoter by the NtrC enhancer-binding pro- segment with the operator-promoter region of the gal operon tein. between the NtrC enhancer site and the promoter ofthe ginA Three principal models have been proposed for enhancer operon. The glnA fragment with the insertion was prepared as action (11-13). In the first, the regulatory protein (or RNA an EcoRI fragment from plasmid pJES371, which was con- polymerase) associates with the DNA at the enhancer site structed as follows. EcoRI linkers were ligated to the 431-bp and then traverses the DNA to the start site for RNA Rsa I fragment from pJES131 (P. Wong and S.K., unpublished synthesis (entry-site or scanning model) (1, 14). In the second work), and this fragment was subcloned into pUC19. A 347-bp model, the enhancer-binding protein facilitates transcription fragment carrying the gal regulatory region (28) was then by initiating a change in DNA structure that is propagated inserted into the Fsp I site at the end ofbinding site 4 for NtrC. from the enhancer site to the promoter (e.g., a site-specific The predicted distance between NtrC and &54-holoenzyme on DNA topoisomerase) (15). In the third model, the enhancer- the EcoRI fragment derived from pJES371 was -390 bp. This bound regulatory protein stimulates transcription by a direct distance was calculated as the length of the gal fragment plus protein-protein interaction with RNA polymerase at the the measured distance between bound NtrC and RNA polymerase on the wild-type fragment pJES125 (e.g., Fig. 3). The publication costs of this article were defrayed in part by page charge Proteins. a-54, the NtrC activator protein [NtrCcon mutant payment. This article must therefore be hereby marked "advertisement" form = NtrC610 (9)], and NtrB [the kinase that phosphory- in accordance with 18 U.S.C. §1734 solely to indicate this fact. lates NtrC (32)] were purified as described (33, 34). 5504 Downloaded by guest on September 29, 2021 Biochemistry: Su et al. Proc. Natl. Acad. Sci. USA 87 (1990) 5505 wild type ginA fragment: NtrC Sites RNA 1 2 3 4 5 1 - gInA I _. _MN Promoter

ginA fragment with insertion: NtrC sites RNA 1 23 4 5 r .ginA Promoter FIG. 1. The glnA enhancer-promoter configurations used to study the action of NtrC. (Upper) A 453-bp restriction fragment carrying the wild-type configuration of enhancer and promoter. The strong binding sites 1 and 2 for NtrC define the enhancer. (Lower) A 782-bp restriction fragment in which the enhancer has been separated from the promoter by the insertion ofa 347-bp segment carrying the operator-promoter region of the gal operon (OE and O1 are the binding sites for the gal repressor protein). Reaction Conditions for the Formation of DNA-Protein that of RNA polymerase (450 kDa). Because free NtrC Complexes. Two buffers were used for reactions to detect dimers have a molecular mass of 110 kDa, the DNA-bound DNA-protein complexes. No significant differences were complex can be presumed to carry two or more NtrC noted between the two buffers in the results of the experi- molecules. Many of the NtrC complexes exhibited a distinct ments. Buffer A contained 50 mM Tris acetate (pH 8.0), 100 bend in the DNA (Fig. 2). From these observations, we mM KOAc, 8 mM Mg(OAc)2, 1 mM dithiothreitol, 27 mM conclude that NtrC probably forms a DNA-bent nucleopro- NH4OAc, and 3.5% (wt/vol) polyethylene glycol. Buffer B tein structure at the enhancer region. contained 40 mM Hepes/KOH (pH 8.0), 100 mM KOAc, 10 NtrC-Mediated Activation of Stable Promoter Complexes by mM MgCl2, 0.1 mM dithiothreitol, and 4% (vol/vol) glycerol. RNA Polymerase. The NtrC protein functions as a positive Proteins were first mixed together and then added to the regulator ofglnA transcription by facilitating the transition of DNA. For the reactions in Table 1, buffer A was used RNA polymerase (cr54-holoenzyme) from a closed to an open together with NtrC (360 nM) and wild-type DNA fragment (34 complex at the promoter site (9). ATP is required directly to nM). When present, RNA polymerase core, C54, and NtrB stimulate formation of open complexes (9). [ATP is also were at 50 nM, 130 nM, and 90 nM, respectively. ATP was required for NtrB-dependent phosphorylation of NtrC (32, added as indicated to 2 mM. After incubation for 20 min at 34)]. Under activation conditions, stable complexes between 37°C, heparin was added to 50 Mg/ml, and 7 min later the RNA polymerase and DNA were detected by electron mi- samples were prepared for electron microscopy. For the croscopy on about 30% ofthe DNA molecules (Fig. 3 c-e and reactions in Table 2, buffer B was used together with NtrC Table 1). The RNA polymerase could be distinguished from (370 nM) and the insertion DNA fragment (70 nM). The NtrC because the transcribing enzyme was more electron- reaction mixtures also contained 110 nM core RNA poly- dense, presumably because ofmore efficient shadowing with merase, 200 nM -54, and 90 nM NtrB. ATP was added as tungsten. As expected for open complexes, formation of indicated in Table 2 to 5 mM. After 10 minm heparin was added stable RNA polymerase-DNA structures was an ATP- to 100 ,ug/ml. Stable associations between proteins (NtrC and dependent reaction (Table 1). A closed complex of RNA a54-holoenzyme) and DNA fragments were monitored by a polymerase is formed in the absence ofNtrC (9); this complex gel retardation assay as well as by electron microscopy. was not observed by electron microscopy, presumably be- Electron Microscopy. After incubation to allow formation cause of its relative instability. Nearly all of the DNA of protein-DNA complexes, the reaction mixtures were molecules with stably bound RNA polymerase also carried diluted 10- to 40-fold into reaction buffer and prepared for NtrC. As judged by measurements of DNA length, the NtrC electron microscopy without fixation, by the polylysine tech- nique (35). Grids were rotary-shadowed with tungsten, and electron microscopy was carried out at a magnification of approximately 35,000. Electron microscopic analysis allowed visualization of NtrC-DNA complexes and stable open complexes ofRNA polymerase at the promoter site, but not unstable closed complexes ofRNA polymerase (see text). RESULTS Interaction of NtrC with Enhancer Site. As judged by DNase I- and methylation-protection assays, the NtrC protein binds to five sites in the glnA promoter region (33) (Fig. 1). When separated from the other sites, the high-affinity sites 1 and 2 alone have properties of a transcriptional enhancer (8). The low-affinity sites 4 and 5 do not appear to be important for activation of transcription (8, 36, 37). We have used electron microscopy to visualize the interaction ofNtrC with its sites under suitable for activation binding conditions FIG. 2. NtrC protein bound to the enhancer site. The DNA of transcription (Fig. 2). As judged by length measurements fragment is the 453-bp wild-type ginA promoter fragment (Fig. 1 of the DNA fragments carrying bound NtrC, the region of Upper). The grid was prepared from a reaction mixture containing DNA occupied by NtrC included all of sites 1 and 2 and at only NtrC protein. As judged by length measurements on the DNA, least the majority of site 3 (data not shown). As noted in Fig. binding sites 1 and 2 and at least the majority of site 3 are associated 3 below, the size ofthe DNA-bound NtrC was comparable to with NtrC. (Bar = 0.1 ,um.) Downloaded by guest on September 29, 2021 5506 Biochemistry: Su et al. Proc. Natl. Acad. Sci. USA 87 (1990) Table 1. DNA-protein complexes of NtrC, RNA polymerase, and wild-type glnA promoter fragments - .., vi ,r;^.'. ~-...' Percent DNA molecules V..,. t Free NtrC Pol + NtrC Possible

.. . 14:4 ATP DNA complexes complexes loops

.'., it , + 5 57 31 5 a . - 13 85 2 0.5 The reaction conditions for formation of DNA-protein complexes ,b are described in Materials and Methods. The reaction mixtures contained NtrC, RNA polymerase oS4-holoenzyme, and NtrB, the kinase that phosphorylates NtrC. "NtrC complexes" are DNA Ii,':.i *.J.K * molecules that carry only NtrC at the enhancer (see Fig. 2). "Pol + NtrC complexes" are DNA molecules carrying both NtrC and specifically bound RNA polymerase (shown in Fig. 3 c-e). The structures labeled "Possible loops" are those in which RNA polymerase at the promoter was adjacent to NtrC bound at the enhancer site (shown in Fig. 3b). Two percent of the DNA fragments in the reaction with ATP carried an RNA polymerase at approximately the promoter location but did not carry NtrC. Four hundred molecules were counted for each reaction. mixture 2). The length of most of the loops was very close to the predicted 390 bp between the enhancer and promoter sites (Fig. 4b). Because ofthe difference in shadowing, many of the looped molecules clearly carried both RNA polymerase and NtrC at the base ofthe loop. Thus, we could conclude that heterologous protein-protein interactions were respon- sible for the loops. If the looped DNA molecules represent productive intermediates in the transcription reaction, the loops should disappear when RNA polymerase transcribes. To test this prediction, we formed open complexes in the presence of ATP, added heparin to prevent formation of new open complexes, and then added the remaining three ribonucleo- side triphosphates to allow transcription. After the transcription reaction, the RNA polymerase complexes and loops were not observed by electron microscopy (Table 2, mixture 4). If the nucleotides were omitted, a large fraction of the polymerase complexes and loops persisted during a 10-min incubation in the presence of heparin (Table 2, mixture 3). From the data of Fig. 4 and Table 2, we conclude that enhancer-bound NtrC activates transcription by a protein- FIG. 3. RNA polymerase (c4-holoenzyme) bound to the pro- protein interaction with RNA polymerase at the promoter site. moter site and NtrC bound at the enhancer. The DNA molecule is the same wild-type glnA promoter fragment shown in Fig. 2. The grids DISCUSSION were prepared from the ATP-containing reaction mixture of Table 1. of NtrC from the Enhancer Site. From the Panels correspond to different species counted in that table: free DNA Activity exper- (a), possible loop (b), and Pol + NtrC complexes (c-e). The molecule iments reported here, we have concluded that enhancer in the upper right corner of c is an NtrC complex. RNA polymerase, function occurs by a direct interaction of DNA-bound NtrC free or bound, had a higher electron density than NtrC, presumably with RNA polymerase, in a reaction that loops out the because it shadowed more efficiently with tungsten. (Bar = 0.1 ,um.) intervening DNA. We have used electron microscopy to visualize looped complexes that associate the enhancer with molecules were located at the enhancer site, and RNA the promoter, demonstrating an interaction over a substantial polymerase occupied the promoter site (Fig. 1) (data not shown). We also observed an apparent association of NtrC Table 2. DNA-protein complexes of NtrC, RNA polymerase, bound at the enhancer site with RNA polymerase bound at and ginA promoter fragments with the distant enhancer the promoter on about 5% of the total DNA molecules (Fig. Additions Percent DNA molecules 3b; possible loops" in Table 1). To define these relatively Reaction Hep- CTP, GTP, Free NtrC Pol + rare interactions more precisely, we introduced a 347-bp mixture ATP arin and UTP DNA only NtrC Loop spacer between the enhancer and the promoter (Fig. 1 - - - 10 85 4 1 Lower). The spacer carried the operator-promoter 1 region 2 + - - 11 53 31 5 for the gal operon. 3 + + - 14 72 12 2 Formation ofDNA Loops Between Enhancer- and Promoter- 4 + + + 13 86 1 <0.2 Bound Proteins. In experiments in which NtrC acted from the more distant enhancer, we found that about 30% of the DNA ATP was added to reaction mixtures 2-4 to allow the formation of molecules carried RNA reaction mix- open complexes, and then heparin (100 pg/ml) was added to mixtures polymerase (Table 2, 3 and 4 to prevent further open complex formation. Thirty seconds ture 2). As expected, the RNA polymerase was localized at later, CTP, GTP, and UTP were added to mixture 4 to allow a single the promoter about 390 bp from the NtrC (data not shown). round oftranscription. After an additional 10 min at 37°C, all reaction More importantly, we observed that 5% of the DNA mole- mixtures were prepared for electron microscopy. Four hundred cules carried a clearly discernable loop (Fig. 4a and Table 2, molecules were counted for mixtures 2-4, and 200 for mixture 1. Downloaded by guest on September 29, 2021 Biochemistry: Su et al. Proc. Natl. Acad. Sci. USA 87 (1990) 5507 a highly dependent on ATP, as is formation ofopen complexes; (ii) the loops disappear when RNA polymerase is allowed to ,.* S.. Aw^. transcribe and is thereby removed from the DNA.

woo., The number of looped structures is about 15% the number .4. ..,E '%VONr.>*s of open complexes. From this observation, we believe that .fl* the enhancer functions in a transient interaction. The proposed reaction pathway is diagrammed in Fig. 5. DNA-bound NtrC facilitates the transition of RNA polymerase from the closed to the open complex and then relaxes to the major equilibrium DNA-protein complex in which most NtrC proteins at the enhancer are separated from RNA polymerase at the promoter. After RNA polymerase transcribes away from the promoter, NtrC can act on a new incoming RNA polymerase. Previous experiments have shown that open complexes formed in the presence of NtrC are competent for transcription after NtrC has been removed by gel filtration (9). In a set of experiments complementary to those reported here, activation by NtrC was retained when the enhancer was located on a separate DNA molecule that was catenated to the promoter DNA (10). This result is inconsistent with scanning or transmission models. In other experiments with the enhancer system described here, we have found that there was no inhibition of NtrC-mediated activation by DNA-bound gal repressors located between the enhancer and the promoter (unpublished work). Given the agreement in interpretation among all of the available experiments, we believe that the DNA-looping pathway for enhancer action shown in Fig. 5 is firmly established. Distant Regulatory Sites in Transcriptional Control. There is increasingly strong evidence that control of most prokaryotic and eukaryotic promoters depends on the action of , . .: :. f t + regulatory proteins from sites that are too distant for protein- * ; _ * * t He . protein contact on linear DNA (6, 7, 38). The use of an array b..,'A*. *.^ * ' .0 m._z*+ <. . '** t t * t Ad .4' of regulatory proteins capable of interacting with each other f *,. al§W X, lB mu IN and with the enzyme machinery at the promoter obviously allows for multiple inputs into the decision of whether or not An_ to transcribe. A summation offunctional interaction domains b on the regulatory proteins (positive or negative) might be the 25 r- B. 4 s, . s key to a transcription switch. For multiple regulatory pur- (u poses, the optimal arrangement might involve an ensemble of a) x 20 1 5- Enhancer Promot,,r E 0 15 - m VF 0 I0 F s ' ' --of NtrC |Pol .0 t ^ ._ ..... ;. _ .._,_ E 5 z I -A - - - 0 _ l_ . Closed Complex 0 -- I I ll r.. I 0 100 200 300 400 500 ATP Loop Size (bp) FIG. 4. DNA loops formed by association of NtrC and RNA Looped Aciivation polymerase-promoter complexes. The DNA molecule is the ginA Intermediate promoter fragment with the insertion (Fig. 1 Lower). (a) Looped molecules. (Bar = 0.1 am.) (b) Distribution of loop lengths. The predicted distance between enhancer and promoter is -390 bp. All looped molecules were measured so that the specific distribution is superimposed on a background of random "flop-overs" in which DNA strands cross on the grid. -w Open Complex and easily measurable distance. In addition to NtrC, the formation oflooped complexes.requiredA_the presence ofRNA FIG. 5. Model for the action of NtrC from the enhancer site. Two polymerase and ATP, indicating that the looped structures or more phosphorylated NtrC molecules are associated with the derived from an association enhancer. The DNA-bound NtrC associates with the RNA polymer- between enhancer-bound NtrC ase present in a closed promoter complex and facilitates isomeriza- and RNA polymerase. We believe that looped structures are tion of the enzyme into the productive open complex. The polymer- intermediates in open-complex formation by RNA polymerase transcribes the ginA__gene, and NtrC remains bound to the ase for two major reasons: (i) formation of DNA loops is enhancer site, available for another round of activation. Downloaded by guest on September 29, 2021 5508 Biochemistry: Su et al. Proc. Natl. Acad. Sci. USA 87 (1990) sites for different regulatory proteins that are close enough to ported by grants from the National Institute of General Medical the promoter for effective collision, but far enough away to Sciences (GM17078 and GM38361). allow DNA-looping without a severe energetic cost in DNA- bending. For each regulatory site, occupancy by a functional 1. Moreau, P., Hen, R., Wasylyk, B., Everrett, R., Gaub, M. & regulatory protein would be the key requirement, and ho- Chambon, P. (1981) Nucleic Acids Res. 9, 6047-6069. mologous interactions between identical regulatory proteins 2. Banerji, J., Rusconi, S. & Schaffner, W. (1981) Cell 27, might serve mainly to increase occupancy. An additional 299-308. 3. Maniatis, T., Goodbourn, S. & Fischer, J. F. (1987) Science requirement for a distant regulator is a means to distinguish 236, 1237-1245. its target promoter from other nearby promoter sites. For 4. Muller, M. M., Gerster, T. & Schaffner, W. (1988) Eur. J. ginA, this is accomplished by the use ofthe a-" form ofRNA Biochem. 176, 485-495. polymerase. 5. Atchison, M. L. (1988) Annu. Rev. Cell Biol. 4, 127-153. The regulatory reaction that we have proposed for NtrC 6. Adhya, S. (1989) Annu. Rev. Gen. 23, 207-250. and &-holoenzyme proceeds solely by means of protein- 7. Gralla, J. D. (1989) Cell 57, 193-195. protein contact. Another type of DNA-looping interaction 8. Reitzer, L. J. & Magasanik, B. (1986) Cell 45, 785-792. might serve to control promoter utilization by constraining or 9. Popham, D. L., Szeto, D., Keener, J. & Kustu, S. (1989) facilitating structural changes in the DNA (e.g., the unwind- Science 243, 629-635. ing necessary for open-complex formation by RNA polymer- 10. Wedel, A., Weiss, D., Popham, D., Droge, P. & Kustu, S. ase). In these cases, the DNA loop between regulatory (1990) Science 248, 486-490. proteins would itself be required, and simple occupancy of 11. Dynan, W. S. & Tjian, R. (1985) Nature (London) 316, 774- 778. binding sites at the base of the loops would not suffice. For 12. Echols, H. (1986) Science 233, 1050-1056. repression of the gal operon, occupancy of both operator 13. Ptashne, M. (1986) Nature (London) 322, 697-701. sites does not provide for effective negative regulation; the 14. Brent, R. & Ptashne, M. (1984) Nature (London) 312, 612-615. additional association of DNA-bound repressors is required 15. Courey, A. J., Plon, S. E. & Wang, J. C. (1986) Cell 45, (28). For this type of interaction, the regulatory sites would 567-574. probably be relatively close to the promoter (as for the gal 16. Echols, H. (1984) BioEssays 1, 148-152. operon), and the regulatory interaction would be unlikely to 17. Gellert, M. & Nash, H. (1987) Nature (London) 325, 401-404. work from substantially more distant sites. Thus, even 18. Echols, H. (1990) in Molecular Mechanisms in DNA Replica- though the loop-mediated interaction ofDNA-bound proteins tion and Recombination, eds. Richardson, C. & Lehman, R. (Liss, New York), pp. 1-31. appears to be the most prevalent mode of controlling tran- 19. Landy, A. (1989) Annu. Rev. Biochem. 58, 913-950. scription, the use of distant sites to regulate promoter utili- 20. Majumdar, A. & Adhya, S. (1984) Proc. Natl. Acad. Sci. USA zation does not necessarily imply an identity of mechanism. 81, 6100-6104. Central Role of Nucleoprotein Interactions in Regulating 21. Dunn, T. M., Hahn, S., Ogden, S. & Schleif, R. F. (1984) Proc. DNA Transactions. Many DNA transactions depend on the Natl. Acad. Sci. USA 81, 5017-5020. association of DNA-bound proteins to generate a multipro- 22. Schleif, R. (1988) Science 240, 127-128. tein regulatory complex (6, 12, 16). In addition to transcrip- 23. Robbins, P., Rio, D. C. & Botchan, M. R. (1986) Mol. Cell. tion, such nucleoprotein structures typically control initia- Biol. 6, 1283-1295. tion of DNA replication and site-specific recombination (12, 24. Griffith, J., Hochschild, A. & Ptashne, M. (1986) Nature (London) 322, 750-752. 16-19). Thus, the association of DNA-bound regulatory 25. Kramer, H., Niemoller, M., Amouyal, M., Revet, B., Wilcken- proteins appears to be the major way in which DNA trans- Bergmann, B. V. & Miller-Hill, B. (1987) EMBO J. 6, 1481- actions are localized and controlled. A useful general term for 1491. such ubiquitous interactions is the specialized nucleoprotein 26. Theveny, B., Bailly, A., Rauch, C., Rauch, M., Delain, E. & structure ("snup") (12). Some snups are transient, whereas Milgrom, E. (1987) Nature (London) 329, 79-81. others pass through a relatively stable phase. The association 27. Amouyal, M., Mortensen, L., Buc, H. & Hammer, K. (1989) between NtrC and RNA polymerase appears to be an exam- Cell 58, 545-551. ple of a transient interaction; the DNA-looped nucleoprotein 28. Mandal, N., Su, W., Haber, R., Adhya, S. & Echols, H. (1990) Genes Dev. 4, 410-418. structure turns over into the transcriptionally active open 29. Oehler, S., Eismann, E. R., Kramer, H. & Muller-Hill, B. complex. For some replication complexes, activation of (1990) EMBO J. 9, 973-979. DNA-unwinding by the initiation helicase requires a more 30. Muller, H.-P., Sogo, J. M. & Schaffner, W. (1989) Cell 58, complicated turnover reaction; heat shock proteins function 767-777. in an ATP-dependent reaction to disassemble the stable 31. Dunaway, M. & Droge, P. (1989) Nature (London) 341, 657- multiprotein assembly (snup) that localizes the helicase at the 659. replication origin (18). For many transcriptional pathways in 32. Ninfa, A. J. & Magasanik, B. (1986) Proc. Natl. Acad. Sci. prokaryotes and eukaryotes, a number ofregulatory proteins USA 83, 5909-5913. must communicate to control the promoter, and a relatively 33. Hirschman, J., Wong, P. K., Sei, K., Keener, J. & Kustu, S. be needed (1985) Proc. Natl. Acad. Sci. USA 82, 7525-7529. stable phase ofthe regulatory interaction might (6, 34. Keener, J. & Kustu, S. (1988) Proc. Natl. Acad. Sci. USA 85, 12, 38). A separate turnover reaction might be operative in 4976-4980. such cases. Whatever the detailed processes, there appears 35. Williams, R. (1977) Proc. Natl. Acad. Sci. USA 74, 2311-2315. to be a fascinating similarity in the general mechanisms for 36. Sasse-Dwight, S. & Gralla, J. D. (1988) Proc. Natl. Acad. Sci. regulating diverse DNA transactions. USA 85, 8934-8938. 37. Reitzer, L. J., Movsas, B. & Magasanik, B. (1989) J. Bact. 171, We thank Richard Eisner for editorial help and Mike Botchan and 5512-5522. Carol Gross for advice about the manuscript. This work was sup- 38. Mitchell, P. J. & Tjian, R. (1989) Science 245, 371-378. Downloaded by guest on September 29, 2021