Single and double loop formation when deoR repressor binds to its natural operator sites

Amouyal, Michèle; Mortensen, L.; Buc, Henri; Hammer, Karin

doi:10.1016/0092-8674(89)90435-2

Cited by 71 publications

(48 citation statements)

References 23 publications

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“…For the initiation of D N A replication and site-specific recombination, there is abundant evidence that communication occurs by the association of DNA-bound proteins in a nucleoprotein structure in which the D N A is looped or wound (Echols 1984(Echols , 1986(Echols , 1990Gellert and Nash 1987;Landy 1989). Direct evidence by electron microscopy has been presented for interaction of DNA-bound proteins in the case of kcI (Griffith et al 1986), LacI (Kr~imer et al 1987), and deo operon repressors (Amouyal et al 1989). However, these demonstrations did not directly correlate D N A looping with repression in a biological system.…”

Section: Transcriptional Control By Association Of Dna-bound Regulatomentioning

confidence: 99%

“…For control of transcription, communication between distant regulatory sites is also thought to involve association between proteins with the consequence of looping the intervening DNA; these interactions may be transient or lead to a more stable complex with other proteins (Irani et al 1983;Dunn et al 1984;Majumdar and Adhya 1984;Echols 1986;Ptashne 1986;Schleif 1988;Adhya 1989;Mitchell and Tjian 1989). Electron microscopic evidence has been presented for DNA-looping interactions involving kcI (Griffith et al 1986), LacI (Kramer et al 1987), and the repressor for the deo operon (Amouyal et al 1989). However, there has not been a study indicating the necessity for loop formation, rather than simple occupancy, for regulation of transcription in vivo.…”

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confidence: 99%

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DNA looping in cellular repression of transcription of the galactose operon.

Mandal¹,

Su²,

Haber³

et al. 1990

Genes Dev.

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Communication between distant DNA sites is a central feature of many DNA transactions. Negative regulation of the galactose (ga/) operon of Escherichia coli requires repressor binding to two operator sites located on opposite sides of the promoter. The proposed mechanism for regulation involves binding of the repressor to both operator sites, followed by a protein-protein association that loops the intervening promoter DNA (double occupancy plus association). To assess these requirements in vivo, we have previously converted ga/operator sites to lac and shown that both operator sites must be occupied by the homologous repressor protein (Lac or Gal) for negative regulation of the ga/operon. We have now addressed more directly the need for proteinprotein association by the use of the converted operator sites and a mutant Lac repressor defective in association of the DNA-binding dimers. We have compared the biological and biochemical activity of two Lac repressors: the wild-type (tetramer) I + form, in which the DNA-binding dimer units are tightly associated; and the mutant I "~ repressor, in which the dimer units do not associate effectively. The I ÷ repressor is an efficient negative regulator of the ga/operon in vivo, but the I "~ mutant is an ineffective repressor. Purified I + repressor efficiently forms DNA loops between operator sites that we have visualized by electron microscopy; the I °~ repressor fails to form DNA loops, although the protein binds effectively to both operator sites. From the clear correlation between looping in vitro and repression in vivo, we conclude that regulation of the ga/operon depends on the association of repressor proteins bound to the two operator sites. Repression is likely to involve a DNA-wound nucleoprotein complex in which RNA polymerase is present but unable to carry out a productive interaction with the promoter sequence.

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Section: Transcriptional Control By Association Of Dna-bound Regulatomentioning

confidence: 99%

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confidence: 99%

DNA looping in cellular repression of transcription of the galactose operon.

Mandal¹,

Su²,

Haber³

et al. 1990

Genes Dev.

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“…More recently, the DNA-looping model for transcriptional regulation has been supported by several more direct types of experiments. DNA-looping interactions between homologous regulatory proteins have been visualized by electron microscopy (24)(25)(26)(27). Recent biological and biochemical experiments have used mutant repressors to correlate these looping interactions directly with negative regulation in vivo in the gal and lac operons (28,29).…”

Section: Introductionmentioning

confidence: 99%

DNA-looping and enhancer activity: association between DNA-bound NtrC activator and RNA polymerase at the bacterial glnA promoter.

Porter

Kustu

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

245

168

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The NtrC protein activates transcription of the ginA operon of enteric bacteria by stimulating the formation of stable "open" complexes by RNA polymerase (oMholoenzyme form). To regulate the ginA promoter, NtrC binds to sites that have the properties of transcriptional enhancers: the sites will function far from the promoter and in an orientation-independent fashion. To investigate the mechanism of enhancer function, we have used electron microscopy to visualize the interactions of purified NtrC and RNA polymerase with their DNA binding sites and with each other. Under conditions that allow the formation of open complexes, about 30% of DNA molecules carry both RNA polymerase and NtrC bound to their specific sites. Of these, about 15% form looped structures in which NtrC and the RNA polymerase-promoter complex are in contact. The length of the looped DNA is that predicted from the spacing that was engineered between the enhancer and the ginA promoter (390 base pairs). As expected for activation intermediates, the looped structures disappear when RNA polymerase is allowed to transcribe the DNA. We conclude that the NtrC enhancer functions by means of a direct association between DNA-bound NtrC and RNA polymerase (DNA-looping model). Association of DNA-bound proteins appears to be the major mechanism by which different types of site-specific DNA transactions are localized and controlled.Enhancer sequences have been defined by their function in activating transcription from relatively long distances in an orientation-independent manner (1, 2). There are many wellcharacterized examples of enhancers controlling eukaryotic cellular and viral genes (3-5). Although initially defined in eukaryotes, regulation of transcription from distant sites has been observed for many prokaryotic promoters (6, 7). The site for positive regulation of the glnA operon of enteric bacteria is a particularly well-defined example of a prokaryotic enhancer (8-10). To investigate the mechanism of enhancer action, we have studied the activation of transcription from the glnA promoter by the NtrC enhancer-binding protein.Three principal models have been proposed for enhancer action (11-13). In the first, the regulatory protein (or RNA polymerase) associates with the DNA at the enhancer site and then traverses the DNA to the start site for RNA synthesis (entry-site or scanning model) (1,14). In the second model, the enhancer-binding protein facilitates transcription by initiating a change in DNA structure that is propagated from the enhancer site to the promoter (e.g., a site-specific DNA topoisomerase) (15). In the third model, the enhancerbound regulatory protein stimulates transcription by a direct protein-protein interaction with RNA polymerase at the promoter site (or with other proteins that contact polymerase) (DNA-looping model) (11-13, 16).The DNA-looping model for enhancer action has been considered attractive for several reasons. The interaction between DNA-bound proteins has been for some time an established principle for contr...

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“…For multioperator prokaryotic regulation, DNA looping was proposed initially to explain the interaction of transcription regulators bound to spatially separated DNA sites (Dunn et al 1984: Majumdar andAdhya 1984). More recently, a looping interaction between homologous regulatory proteins has been visualized directly by electron microscopy (Griffith et al 1986;Kr/imer et al 1987;Amouyal et al 1989;Mandal et al 1990). Recent biological and biochemical experiments with mutant repressors have correlated DNA-looping interactions with negative regulation in vivo in the gal and lac operons (Mandal et al 1990;Oehler et al 1990).…”

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confidence: 99%

DNA looping between sites for transcriptional activation: self-association of DNA-bound Sp1.

Su¹,

Jackson²,

Tjian³

et al. 1991

Genes Dev.

331

203

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The Spl protein activates transcription from many eukaryotic promoters. Spl can act in vivo from enhancer sites that are distal to the promoter and exhibit synergistic interaction with promoter-proximal binding sites. To investigate possible protein-protein interactions between DNA-bound Spl molecules, we have used electron microscopy to visualize the DNA-protein complexes. At the SV40 promoter, we observed the expected localized interaction at the Spl sites; in addition, we found that DNA-bound Spl served to associate two or more DNA molecules. At a modified thymidine kinase promoter, we observed a localized interaction at each of two binding locations that were separated by 1.8 kbp; in addition, we noted a substantial fraction of DNA molecules in which the distant binding regions were joined by a DNA loop. As judged by studies with mutant Spl proteins, the distant interactions depended on the glutamine-rich regions of Spl required for transcriptional activation. We conclude that DNA-bound Spl can self-associate, bringing together distant DNA segments. From the correlation between DNA looping in vitro and synergistic activation of the modified thymidine kinase promoter shown previously in vivo, we suggest that Spl exerts its transcriptional synergism by a direct protein-protein association that loops the intervening DNA. Our experiments support the DNA-looping model for the function of transcriptional enhancers.[Key Words: Spl protein; DNA looping; DNA binding; transcription]Received December 26, 1990; revised version accepted January 23, 1991.The controlled initiation of transcription in prokaryotes and eukaryotes depends on the action of regulatory proteins from sites that are too distant for a direct interaction with promoter-bound proteins on linear DNA. Three principal models have been proposed for positive regulation from distant (enhancer) sites (Dynan and Tjian 1985;Echols 1986;Ptashne 1986). In the first, the regulatory protein (or RNA polymerase) associates with the DNA at the enhancer site and then traverses the DNA to the promoter site (scanning model). In the second, the enhancer-binding protein initiates a change in DNA structure that is propagated from the enhancer to the promoter, thereby activating transcription (structural transmission model). In the third, the enhancerbound regulatory protein activates transcription by a direct protein-protein interaction with RNA polymerase at the promoter (or other proteins that contact polymerase) (DNA-looping or nucleoprotein model).DNA looping is currently favored as the most likely mechanism for distant regulatory interactions. The interaction between DNA-bound proteins has been firmly established as the central structural feature for regulated 3Present address:

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Single and double loop formation when deoR repressor binds to its natural operator sites

Cited by 71 publications

References 23 publications

DNA looping in cellular repression of transcription of the galactose operon.

DNA looping in cellular repression of transcription of the galactose operon.

DNA-looping and enhancer activity: association between DNA-bound NtrC activator and RNA polymerase at the bacterial glnA promoter.

DNA looping between sites for transcriptional activation: self-association of DNA-bound Sp1.

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