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

Mandal, Nripendranath; Su, Wen; Haber, Roberta; Adhya, Sankar; Echols, Harrison

doi:10.1101/gad.4.3.410

Cited by 84 publications

(70 citation statements)

References 41 publications

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“…LacI forms tetramers by flexible C-terminal heptad repeats. The latter loop did not show a kinked DNA by electron microscopy (Mandal et al 1990). Similarly, tetramerization of hybrid GalR via the LacI flexible Cterminal heptad repeats results in poor repression compared to dimeric GalR in vivo .…”

Section: Discussionmentioning

confidence: 87%

DNA trajectory in the Gal repressosome

Semsey¹,

Tolstorukov²,

Virnik³

et al. 2004

Genes Dev.

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Section: Discussionmentioning

confidence: 87%

DNA trajectory in the Gal repressosome

Semsey¹,

Tolstorukov²,

Virnik³

et al. 2004

Genes Dev.

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“…Both events would presumably create an effective barrier to diffusion of supercoils generated by transcription (10,27). Although O, GalR, and LacI are each capable of forming DNA loops (28)(29)(30)(31)(32), it is unlikely that either DNA-looping or DNA-pairing events explain the stimulation of DNA supercoiling mediated by open complexes of E. coli RNA polymerase (Table 1) or by the combination of O and EcoRI-Gln-111 proteins (Fig. 5D).…”

Section: Discussionmentioning

confidence: 99%

Potent stimulation of transcription-coupled DNA supercoiling by sequence-specific DNA-binding proteins

Leng

McMacken

2002

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

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Transcription by RNA polymerase can stimulate localized DNA supercoiling in Escherichia coli. In vivo, there is extensive experimental support for a ''twin-domain'' model in which positive DNA supercoils are generated ahead of a translocating RNA polymerase complex and negative supercoils are formed behind it. Negative supercoils accumulate in the template DNA because the positive supercoils are preferentially removed by cellular topoisomerase action. Yet, in vitro, clear and convincing support for the twindomain mechanism has been lacking. In this article, we reconcile this inconsistency by showing that, in a defined in vitro system with plasmid DNA templates, a variety of sequence-specific DNAbinding proteins, such as the bacteriophage O replication initiator or the E. coli lactose or galactose repressors, strikingly stimulate transcription-coupled DNA supercoiling. We demonstrate further that this stimulation requires the presence in the DNA template of a recognition sequence for the relevant DNA-binding protein and depends on the production of long RNA chains by an RNA polymerase. Our data are most consistent with a model in which specific DNA-binding proteins facilitate a twin-domain mechanism to enhance DNA supercoiling during transcription. More precisely, we suggest that some nucleoprotein complexes, perhaps those that contain sharply bent DNA, can form barriers that impede the diffusion and merger of independent chromosomal supercoil domains. Localization of DNA supercoils by nucleoprotein complexes may serve as a general mechanism for modulating DNA transactions that are sensitive to DNA superhelicity.A number of DNA transactions in Escherichia coli are greatly facilitated by negative supercoiling of the bacterial genome (1). For example, opening of the DNA duplex during initiation of transcription at certain promoters and during initiation of DNA replication at the chromosomal origin is energetically favored when the DNA template is negatively supercoiled (see refs. 1 and 2 for reviews). The global DNA supercoiling level in E. coli is principally set through the opposing actions of DNA gyrase and DNA topoisomerase I (Topo I) (3, 4), although recent findings indicate that DNA Topo IV also participates in the relaxation of negatively supercoiled DNA (5).Several studies have demonstrated that the process of transcription of cellular DNA can enhance negative DNA supercoiling locally (3, 6-8). Liu and Wang formulated the ''twinsupercoiled-domain'' model (9) to explain this effect of transcription on DNA supercoiling. Extensive experimental support has been obtained in vivo (reviewed in refs. 1, 10, and 11) for the idea that positive DNA supercoils are generated in front of an advancing transcription complex while an equivalent number of negative DNA supercoils are formed behind it. Formation of these twin supercoiled domains occurs only when the translocating RNA polymerase and associated macromolecules are unable to rotate freely about the DNA helical axis. Under this condition, the template DNA rotates i...

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“…In the experiments of urified Proteins. Wild-type gal repressor (GalR+) and wild-type (LacI+) and mutant (LacIad) lac repressors were purified as described (6,13). CRP was purified by FPLC (Pharmacia) from an E. coli strain carrying the crp+ gene on a multicopy plasmid, pHA5 (gift of S. Ryu and S. Garges).…”

Section: Methodsmentioning

confidence: 99%

“…DNA looping participates in repression as well as in activation of transcription initiation. In the gal operon of Escherichia coli, repression of transcription from two partially overlapping promoters, P1 and P2, requires DNA looping generated by interaction of gal repressor (GaIR protein) molecules bound to two operators, OE and OI, which encompass the promoters and are separated by 11 helical turns of B-DNA (2)(3)(4)(5)(6). As presaged, a repressor can potentially inhibit transcription from a promoter by preventing (i) RNA polymerase (or an activator protein) from binding, (ii) RNA polymerase open complex formation, (iii) formation of the initial phosphodiester bonds, or (iv) promoter clearance by RNA polymerase (7,8).…”

mentioning

confidence: 99%

Control of gal transcription through DNA looping: inhibition of the initial transcribing complex.

Choy

1992

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

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134

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(refs. 7 and 9; A. Majumdar and S.A., unpublished work). These results indicate that GaIR acts at one of the latter three steps described above. We have studied the involvement ofDNA looping and the precise level of repressor action in vitro. Interestingly, the gal operon can be repressed also by lac repressor (LacI protein), provided that both the bipartite gal operators are replaced by lac operators (5). This allowed us to investigate the mechanism of repression of P1, an activator-dependent promoter, and P2, a factor-independent promoter, simultaneously by using both GaIR and Lacd repressors.Since topologically superhelical conformations of DNA have been shown to enhance the interactions between LacR and other transcriptional regulatory proteins to their cognate DNA sites in the formation of DNA loops (10-12), the effect of repressors on gal transcription was studied by using supercoiled "minicircle" DNA templates (unpublished work). These DNA minicircles, containing only gal and no other promoters, greatly facilitated investigating the mechanism ofrepressor action on the synthesis ofboth aborted and full-length transcripts from the gal promoters in the same assay. We report that (i) repression of the gal operon in vitro requires an interaction between repressors bound to two operators; (ii) repression occurs at a step prior to formation of the first phosphodiester bond; and (iii) while Lacd represses both P1 and P2, repression by GaIR, as opposed to the in vivo result, is incomplete for P1 and totally ineffective for P2. MATERIALS AND METHODSPlasmid and Bacteral Strains. Construction and functional elements ofthe plasmids used to generate supercoiled "minicircles" in vivo will be reported elsewhere. Briefly, the parental plasmid carried a multiple cloning site into which the gal promoter segment was inserted. This was followed by a transcription terminator. The promoter-terminator region was located on the plasmid between the A phage attachment site, attP, and the corresponding bacterial site, attB. DNA minicircles carrying the gal promoter followed by the transcription terminator were generated by site-specific recombination between the attP and attB sites in vivo in a host (SA1751) that provided the A integrase and the host integrase factor, IHF. The minicircles were extracted and purified by gel electrophoresis (unpublished work).pSA508, the parental plasmid, contained no promoter at the multiple cloning site. pSA509 contained a 288-base-pair (bp) segment of gal promoter (-197 to +91) cloned between the EcoRI and Pst I sites ofpSA508 (2). pSA510 was identical to pSA509 except that both gal operators, OE and OI, were replaced with the consensus lac operator sequence, 5'-TTGTGAGCGCTCACAA-3' (5). pSA511 and pSA512 were also identical to pSA509 except that the internal operator (Or) or the external operator (OE) were replaced with the lac operator sequence, respectively. In the experiments of urified Proteins. Wild-type gal repressor (GalR+) and wild-type (LacI+) and mutant (LacIad) lac represso...

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

Cited by 84 publications

References 41 publications

DNA trajectory in the Gal repressosome

DNA trajectory in the Gal repressosome

Potent stimulation of transcription-coupled DNA supercoiling by sequence-specific DNA-binding proteins

Control of gal transcription through DNA looping: inhibition of the initial transcribing complex.

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