Structural and Functional Studies of an Intermediate on the Pathway to Operator Binding by Escherichia coli MetJ

He, Yanping; Garvie, Colin W.; Elworthy, Stone; Manfield, Iain W.; McNally, Teresa; Lawrenson, Isobel D.; Phillips, Simon E. V.; Stockley, Peter G.

doi:10.1016/s0022-2836(02)00423-0

Cited by 17 publications

(23 citation statements)

References 39 publications

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“…4). Cooperative interactions between ParG dimers bound to neighboring boxes likely generate a nucleoprotein complex that is sufficiently stable to be detected experimentally, as has been noted with other RHH repressors (21,42). Cooperative interactions in the assembly of ParG on O F are most obvious in the case of site D. Instead of the canonical 5Ј-ACTC-3Ј motifs, this site consists of two 5Ј-ACTT-3Ј motifs separated by a 4-bp spacer that, unlike the AT-rich spacer in other sites, includes a C residue (Fig.…”

Section: Discussionmentioning

confidence: 76%

“…The intensity of each band was determined using Gel-Pro Analyzer 3.1 (Media Cybernetics) software after subtracting background from a blank area adjacent to each sample band. ParG affinity for different DNA sites was determined as the apparent equilibrium dissociation constant K app (defined as the concentration of protein required to generate 50% occupancy), which was estimated from binding curves, similar to previous analysis of the MetJ RHH protein (21). The term K app is used because protein concentration and activity (the effective concentration) are different (29) due to the use of a competitor in binding reactions (24,25).…”

Section: Methodsmentioning

confidence: 99%

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Recruitment of the ParG Segregation Protein to Different Affinity DNA Sites

et al. 2009

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The segrosome is the nucleoprotein complex that mediates accurate plasmid segregation. In addition to its multifunctional role in segrosome assembly, the ParG protein of multiresistance plasmid TP228 is a transcriptional repressor of the parFG partition genes. ParG is a homodimeric DNA binding protein, with C-terminal regions that interlock into a ribbon-helix-helix fold. Antiparallel ␤-strands in this fold are pre-

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Section: Discussionmentioning

confidence: 76%

Section: Methodsmentioning

confidence: 99%

Recruitment of the ParG Segregation Protein to Different Affinity DNA Sites

et al. 2009

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“…protein-protein co-operativity in regulation was also investigated using a MetJ mutant (Q44K) characterized in vitro as having reduced co-operativity in operator saturation [30].…”

Section: Introductionmentioning

confidence: 99%

Transcript analysis reveals an extended regulon and the importance of protein–protein co-operativity for the Escherichia coli methionine repressor

Marincs

Manfield

Stead

et al. 2006

Biochemical Journal

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We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene, metJ, on the Escherichia coli transcriptome. We assayed the effects in the knockout strain of supplying wild-type or mutant MetJ repressors from an expression plasmid, thus establishing a rapid assay for in vivo effects of mutations characterized previously in vitro. Repression is largely restricted to known genes involved in the biosynthesis and uptake of methionine. However, we identified a number of additional genes that are significantly up-regulated in the absence of repressor. Sequence analysis of the 5 promoter regions of these genes identified plausible matches to met-box sequences for three of these, and subsequent electrophoretic mobility-shift assay analysis showed that for two such loci their repressor affinity is higher than or comparable with the known metB operator, suggesting that they are directly regulated. This can be rationalized for one of the loci, folE, by the metabolic role of its encoded enzyme; however, the links to the other regulated loci are unclear, suggesting both an extension to the known met regulon and additional complexity to the role of the repressor. The plasmid gene replacement system has been used to examine the importance of protein-protein co-operativity in operator saturation using the structurally characterized mutant repressor, Q44K. In vivo, there are detectable reductions in the levels of regulation observed, demonstrating the importance of balancing protein-protein and protein-DNA affinity.

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“…It is possible, but unlikely, that the natural sequence variations alter the interaction between adjacent MetJ dimers in the complex. There is a long history of using the consensus sequence as well as natural operators to study MetJ binding (6,7,9,(21)(22)(23).…”

Section: Discussionmentioning

confidence: 99%

“…Thus, it is possible that in vivo repression is only achieved by the binding of four to five MetJ dimers, and that this tight binding mode is different from the weak binding of two to three dimers. While most of the in vitro work has been done with synthetic minimal operators containing only two metboxes, sometimes embedded within longer DNA fragments, the metboxes used are always composed of repeats of the consensus sequence (6,7,9,(21)(22)(23). Consequently, MetJ has an unnaturally high affinity for them.…”

Section: Discussionmentioning

confidence: 99%

Structural Basis for the Differential Regulation of DNA by the Methionine Repressor MetJ

Augustus

Reardon

Heller

et al. 2006

Journal of Biological Chemistry

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The Met regulon in Escherichia coli encodes several proteins responsible for the biosynthesis of methionine. Regulation of the expression of most of these proteins is governed by the methionine repressor protein MetJ and its co-repressor, the methionine derivative S-adenosylmethionine. Genes controlled by MetJ contain from two to five sequential copies of a homologous 8-bp sequence called the metbox. A crystal structure for one of the complexes, the repressor tetramer bound to two metboxes, has been reported (Somers, W. S., and S. E. Phillips (1992) Nature 359, 387-393), but little structural work on the larger assemblies has been done presumably because of the difficulties in crystallization and the variability in the number and sequences of metboxes for the various genes. Small angle neutron scattering was used to study complexes of MetJ and S-adenosylmethionine with double-stranded DNA containing two, three, and five metboxes. Our results demonstrate that the crystal structure of the two-metbox complex is not the native solution conformation of the complex. Instead, the system adopts a less compact conformation in which there is decreased interaction between the adjacent MetJ dimers. Models built of the higher order complexes from the scattering data show that the three-metbox complex is organized much like the two-metbox complex. However, the five-metbox complex differs significantly from the smaller complexes, providing much closer packing of the adjacent MetJ dimers and allowing additional contacts not available in the crystal structure. The results suggest that there is a structural basis for the differences observed in the regulatory effectiveness of MetJ for the various genes of the Met regulon.Transcriptional levels of several proteins in Escherichia coli that are involved in the biosynthesis and transport of methionine are under the common control of the met repressor protein MetJ (1, 2). MetJ is a 12-kDa protein that is reported to form a homodimer in its native state (3). Repressor activity results from MetJ binding to specific 8-bp DNA sequences called metboxes, located in the promoter regions of genes in the Met regulon. Although MetJ selectively binds metbox sequences alone, its affinity for metbox DNA is enhanced severalfold by its co-repressor, S-adenosylmethionine (SAM), 3 an end product of methionine biosynthesis (4 -7). The MetJ protein dimer has been crystallized in the apo and holo forms and in complex with metbox DNA as a tetramer (8, 9). The structure of the protein dimer remains essentially invariant in the three crystals and shows that MetJ is a member of the ribbon-helix-helix family of DNAbinding proteins that bind DNA through a two-stranded ␤-ribbon (9).At least 12 genes, scattered throughout the E. coli chromosome, are repressed by MetJ. Multiple MetJ dimers bind to operator sequences that contain two to five contiguous metboxes. A minimum of two tandem metboxes is required for efficient MetJ binding in vitro and repression of transcription in vivo (6). Adjacent MetJ dimers can int...

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Structural and Functional Studies of an Intermediate on the Pathway to Operator Binding by Escherichia coli MetJ

Cited by 17 publications

References 39 publications

Recruitment of the ParG Segregation Protein to Different Affinity DNA Sites

Recruitment of the ParG Segregation Protein to Different Affinity DNA Sites

Transcript analysis reveals an extended regulon and the importance of protein–protein co-operativity for the Escherichia coli methionine repressor

Structural Basis for the Differential Regulation of DNA by the Methionine Repressor MetJ

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