Mutations that reduce expression from the P2 promoter of the Escherichia coli galactose operon

Bingham, Alistair H.A.; Ponnambalam, Sreenivasan; Chan, Bernard; Busby, Stephen J. W.

doi:10.1016/0378-1119(86)90268-4

Cited by 65 publications

(71 citation statements)

References 23 publications

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“…, in which the P2 promoter was inactivated by a single nucleotide change (10,11), almost 100% of the transcription was initiated from P1, as expected (Fig. 3A).…”

Section: Race Primer Extension Assaysupporting

confidence: 84%

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Quantification of the galactose-operon mRNAs 5 bases different in their 5'-ends

Wang²,

Jeon³

et al. 2010

BMB Reports

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Fig. 1. A schematic presentation of the galactose operon. The -10 regions of the P1 and P2 promoters are shown in boxes. The transcription initiation sites are indicated by arrows and underlined. CRP points to its DNA binding site. OE and OI designate the operators where GalR binds. HU protein binds to hbs. The transcription initiation site of the P1 promoter is assigned as +1, Nucleotide positions of the operon, thus, have been assigned relative to the P1 initiation site.

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“…, in which the P2 promoter was inactivated by a single nucleotide change (10,11), almost 100% of the transcription was initiated from P1, as expected (Fig. 3A).…”

Section: Race Primer Extension Assaysupporting

confidence: 84%

“…3A). In another mutant strain, MG1655kanP1 -P2 + , in which the P1 promoter has been inactivated by a single nucleotide change (10,11), most of the gal transcription was initiated http://bmbreports.org from P2 (Fig. 3A).…”

Section: Race Primer Extension Assaymentioning

confidence: 99%

Quantification of the galactose-operon mRNAs 5 bases different in their 5'-ends

Wang²,

Jeon³

et al. 2010

BMB Reports

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“…As shown schematically in Figure 1B, two chromosomal fusions were constructed in the reporter strain (DM0022). In one of these, the gal promoter was mutated to inactivate P2 transcription while retaining P1 transcription (Bingham et al 1986) and fused to the lacZ gene such that O I was deleted. This fusion, in which lacZ transcription from P1 is repressed by GalR binding to O E , was used to measure GalR-binding activity.…”

Section: Genetic Screen For Galr Mutants Defective In Dna Loopingmentioning

confidence: 99%

“…This fusion, in which lacZ transcription from P1 is repressed by GalR binding to O E , was used to measure GalR-binding activity. To measure loop formation, the gal promoter in a second fusion was mutated so that transcription occurred only from P2 (Bingham et al 1986) and joined to the gusA reporter gene such that O I was retained. The efficacy of the promoter mutations in completely inactivating P1 or P2 while retaining transcription from the other promoter was verified by measuring RNA synthesis in vivo by primer extension.…”

Section: Genetic Screen For Galr Mutants Defective In Dna Loopingmentioning

confidence: 99%

GalR mutants defective in repressosome formation

Geanacopoulos¹,

Vasmatzis²,

Lewis³

et al. 1999

Genes & Development

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Transcription repression of the galactose operon of Escherichia coli requires (1) the binding of the GalR repressor to tandem operators flanking the promoters, (2) the binding of histone-like protein, HU, to a site between the GalR-binding sites, and (3) negatively supercoiled DNA. Under these conditions, protein-protein interactions mediate the formation of a nucleoprotein complex in the form of a DNA loop, which we have termed a repressosome. To analyze the structure of the repressosome, we have screened and isolated galR mutants in which single amino acid substitutions in GalR lead to defects in loop formation while the protein's operator-binding activity is retained. The mutant proteins were purified and their properties confirmed in vitro. We verified that in the case of the two stronger mutations, the proteins had secondary structures that were identical to that of wild-type GalR as reflected by circular dichroism spectroscopy. Homology-based modeling of GalR by use of the crystal structures of PurR and LacI has enabled us to place the three sites of mutation in a structural context. They occur in the carboxy-terminal subdomain of the GalR core, are surface exposed, and, therefore, may be involved in protein-protein interactions. On the basis of our model of GalR and its structural alignment with LacI and PurR, we have identified additional residues, the substitution of which leads to a specific defect in repression by looping. The effects of the mutations are the same in the presence of HMG-17, a eukaryotic protein unrelated to HU, which can also mediate GalR-dependent repression of the gal promoter. This observation suggests that the mutations define sites of GalR-GalR interaction rather than HU-GalR interaction in the repressosome.[Key Words: Repressosome; transcription; galR mutants; protein-protein interactions; DNA looping; GalR; repression] Received October 13, 1998; revised version accepted April 6, 1999.DNA looping is a general phenomenon in the control of transcription in which two or more DNA-binding proteins recognize DNA sequences flanking a promoter and, by contacting each other, cause a reversible transition to a nucleoprotein complex in which the intervening DNA is looped out and the promoter activity is altered (Adhya 1989; Martin et al. 1990). The relative simplicity of these complexes in prokaryotes compared with their counterparts in eukaryotes has facilitated the investigation by genetic and physicochemical methods of questions critical to the understanding of regulation by DNA looping. Some of these questions are structural in nature: What are the protein-protein and protein-DNA contacts that stabilize these loops? What are the symmetries, if any, of the protein complexes? Other questions are framed in terms of thermodynamics and seek to understand the origin of the negative-free energy of the looped structure.Critical mechanistic questions include how loop formation modulates transcription activity and by what means the stability of the loop is sensitive to environmental conditions.W...

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“…In recent work, we and others have measured the effects of over 60 different point mutations in the gal operon promoter region [3][4][5][6][7][8]. Surprisingly, with one exception, none of the mutations cause drastic reductions in expression from the gal promoter region.…”

Section: Introductionmentioning

confidence: 99%

Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters Interactions with RNA polymerase and the cyclic AMP receptor protein

1987

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We report in vitro studies of the interactions between purified E. coli RNA polymerase and DNA from the regulatory region of the E. coli galactose operon which carries a point mutation that simultaneously stops transcription initiation at the two normal start points, $1 and $2. In the presence of this point mutation, transcription initiates at a third start point 14/15 bp downstream of $1, showing that inactivation of the two normally active promoters, P1 and P2, unmasks a third weaker promoter, P3. Transcription initiation in the gal operon is normally regulated by the cyclic AMP receptor protein, CRP, that binds to the gal regulatory region and switches transcription from P2 to PI. With the point mutation, CRP binding switches transcription from P3 to P1, although the formation of transcriptionally competent complexes at P1 is very slow. The results are discussed with respect to the mechanism of transcription activation by the CRP factor and the similarities between the regulatory regions of the galactose and lactose operons.

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Mutations that reduce expression from the P2 promoter of the Escherichia coli galactose operon

Cited by 65 publications

References 23 publications

Quantification of the galactose-operon mRNAs 5 bases different in their 5'-ends

Quantification of the galactose-operon mRNAs 5 bases different in their 5'-ends

GalR mutants defective in repressosome formation

Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters Interactions with RNA polymerase and the cyclic AMP receptor protein

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