A control element within a structural gene: The gal operon of Escherichia coli

Irani, M; Orosz, László; Adhya, Sankar

doi:10.1016/0092-8674(83)90064-8

Cited by 205 publications

(127 citation statements)

References 18 publications

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“…That the NADP effect is primarily on the inhibition of transcription rather than on DNA binding by CcpA may, in part, be comparable with the role of D-galactose in the induction of the E. coli gal operon. Transcription from the galP1 promoter is repressed by GalR bound to its cognate operator (O E ), and the repression is relieved by the presence of D-galactose (39,40). The role of the inducer in derepression may not be to reduce the affinity of GalR for the operator but to allosterically change GalR so that it no longer interacts with the ␣-subunit of RNAP to block transcription (41,42).…”

Section: Discussionmentioning

confidence: 99%

NADP, corepressor for the Bacillus catabolite control protein CcpA

Kim

Voskuil

Chambliss

1998

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

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Expression of the ␣-amylase gene (amyE) of Bacillus subtilis is subject to CcpA (catabolite control protein A)-mediated catabolite repression, a global regulatory mechanism in Bacillus and other Gram-positive bacteria. To determine effectors of CcpA, we tested the ability of glycolytic metabolites, nucleotides, and cofactors to affect CcpA binding to the amyE operator, amyO. Those that stimulated the DNA-binding affinity of CcpA were tested for their effect on transcription. HPr-P (Ser-46), proposed as an effector of CcpA, also was tested. In DNase I footprint assays, the affinity of CcpA for amyO was stimulated 2-fold by fructose-1,6-diphosphate (FDP), 1.5-fold by oxidized or reduced forms of NADP, and 10-fold by HPr-P (Ser-46). However, the triple combinations, CcpA͞NADP͞HPr-P (Ser-46) and CcpA͞FDP͞ HPr-P (Ser-46) synergistically stimulated DNA-binding affinity by 120-and 300-fold, respectively. NADP added to CcpA specifically stimulated transcription inhibition of the amyE promoter by 120-fold. CcpA combined with HPr (Ser-46) inhibited transcription from the amyE promoter, but it also inhibited several control promoters. FDP did not stimulate transcription inhibition by CcpA nor did the triple combinations. The finding that NADP had little effect on CcpA DNA binding but increased the ability of CcpA to inhibit transcription suggests that catabolite repression is not simply caused by CcpA binding amyO but rather a result of interactions with the transcription machinery enhanced by NADP.

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Section: Discussionmentioning

confidence: 99%

NADP, corepressor for the Bacillus catabolite control protein CcpA

Kim

Voskuil

Chambliss

1998

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

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“…Thus, we believe that negative regulation by the classical model is likely to be rare. Previous work has led to the proposal that proteins bound to noncontiguous regulatory sites may communicate with each other by protein-protein association, looping out the intervening D N A (Irani et al 1983;Dunn et al 1984;Majumdar and Adhya 1984). 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).…”

Section: Transcriptional Control By Association Of Dna-bound Regulatomentioning

confidence: 99%

“…In the formation of these higher order structures, an essential step is a communication between proteins bound to spatially separated DNA sites. 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).…”

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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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“…Dimers of GalR bind to the O I and O E sites separated by 113 bp which encompass the partially overlapping P G1 and P G2 promoters (Irani et al 1983). Looping by GalR was hypothesized to explain the molecular mechanism that defined the dependence of P G1 and P G2 promoter activity from O I and O E .…”

Section: Dna Unwinding (Negative Supercoiling) Is a Key Regulator Of mentioning

confidence: 99%

Supercoiling biases the formation of loops involved in gene regulation

Finzi

Dunlap

2016

Biophys Rev

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The function of DNA as a repository of genetic information is well-known. The post-genomic effort is to understand how this information-containing filament is chaperoned to manage its compaction and topological states. Indeed, the activities of enzymes that transcribe, replicate, or repair DNA are regulated to a large degree by access. Proteins that act at a distance along the filament by binding at one site and contacting another site, perhaps as part of a bigger complex, create loops that constitute topological domains and influence regulation. DNA loops and plectonemes are not necessarily spontaneous, especially large loops under tension for which high energy is required to bring their ends together, or small loops that require accessory proteins to facilitate DNA bending. However, the torsion in stiff filaments such as DNA dramatically modulates the topology, driving it from extended and genetically accessible to more looped and compact, genetically secured forms. Furthermore, there are accessory factors that bias the response of the DNA filament to supercoiling. For example, small molecules like polyamines, which neutralize the negative charge repulsions along the phosphate backbone, enhance flexibility and promote writhe over twist in response to torsion. Such increased flexibility likely pushes the topological equilibrium from twist toward writhe at tensions thought to exist in vivo. A predictable corollary is that stiffening DNA antagonizes looping and bending. Certain sequences are known to be more or less flexible or to exhibit curvature, and this may affect interactions with binding proteins. In vivo all of these factors operate simultaneously on DNA that is generally negatively supercoiled to some degree. Therefore, in order to better understand gene regulation that involves protein-mediated DNA loops, it is critical to understand the thermodynamics and kinetics of looping in DNA that is under tension, negatively supercoiled, and perhaps exposed to molecules that alter elasticity. Recent experiments quantitatively reveal how much negatively supercoiling DNA lowers the free energy of looping, possibly biasing the operation of genetic switches.

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A control element within a structural gene: The gal operon of Escherichia coli

Cited by 205 publications

References 18 publications

NADP, corepressor for the Bacillus catabolite control protein CcpA

NADP, corepressor for the Bacillus catabolite control protein CcpA

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

Supercoiling biases the formation of loops involved in gene regulation

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