Kinetic analysis of operator binding by the <i>E. coli</i> methionine repressor highlights the role(s) of electrostatic interactions

Lawrenson, Isobel D.; Stockley, Peter G.

doi:10.1016/s0014-5793(04)00336-9

Cited by 12 publications

(13 citation statements)

References 27 publications

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“…Other evidence for nonspecific MetJ/DNA association comes from surface plasmon resonance kinetic studies with varying sizes of DNA fragments, which show that MetJ can find a target site faster when it is embedded within longer pieces of DNA (18). This work, along with our present study, suggests a mechanism for the enhancement of MetJ's ability to find its specific DNA target genes.…”

Section: Discussionsupporting

confidence: 57%

MetJ repressor interactions with DNA probed by in-cell NMR

Augustus¹,

Reardon²,

Spicer

2009

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

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Atomic level characterization of proteins and other macromolecules in the living cell is challenging. Recent advances in NMR instrumentation and methods, however, have enabled in-cell studies with prospects for multidimensional spectral characterization of individual macromolecular components. We present NMR data on the in-cell behavior of the MetJ repressor from Escherichia coli, a protein that regulates the expression of genes involved in methionine biosynthesis. NMR studies of whole cells along with corresponding studies in cell lysates and in vitro preparations of the pure protein give clear evidence for extensive nonspecific interactions with genomic DNA. These interactions can provide an efficient mechanism for searching out target sequences by reducing the dependence on 3-dimensional diffusion through the crowded cellular environment. DNA provides the track for MetJ to negotiate the obstacles inherent in cells and facilitates locating and binding specific repression sites, allowing for timely control of methionine biosynthesis.DNA-protein interactions ͉ gene regulation ͉ met repressor ͉ methionine regulon ͉ nonspecific DNA T he environment in which proteins and nucleic acids function within a cell is well known to be crowded and complex (1). Understanding the influence of the numerous large and small molecule components on the biophysical and functional properties of these macromolecules in cellular environments, however, is extremely difficult (2, 3). For example, the detailed mechanism of gene regulation is of great interest, but the study of these interactions within living cells has always been a challenge.The transcriptional repressor MetJ controls the expression in Escherichia coli of the Met regulon, which is composed of at least 12 genes scattered at 7 sites around the genome (Fig. 1). These genes code for proteins that are involved in methionine biosynthesis and transport (4, 5). The promoters of these genes contain from 2 to 5 tandem repeats of the MetJ binding site, which are called metboxes and have the consensus sequence AGACGTCT (6). When MetJ is activated by S-adenosylmethionine (SAM) it binds tightly to the metboxes and shuts down transcription (7,8). The different genes repressed by MetJ not only have a variable number of metboxes but also variable sequences. MetJ is thus capable of specific binding to several variations of the consensus sequence in vivo and fine-tuning its repression of each gene (4, 9). NMR spectroscopy has recently been shown to provide a valuable strategy for studying proteins in living cells (10-13). This in-cell spectroscopy gives atomic level fingerprints and even 3-dimensional spectral data that are used to assign resonances and characterize features of individual components in the cellular milieu (12). The method has been used to show ligand binding (14), protein-protein interactions (15) and in at least 1 case, a gain of structure presumably as a result of molecular crowding inside the cell (13). Here, we report NMR results on both in-cell and cell lysate studie...

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Section: Discussionsupporting

confidence: 57%

MetJ repressor interactions with DNA probed by in-cell NMR

Augustus¹,

Reardon²,

Spicer

2009

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

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“…The 5Ј phosphate group of the A site-bound adenosine nucleotide interacts with the peptide backbone amide of residue Lys-31. Although the closest approach of the side chain N group of residue Lys-31 to the RNA is Ϸ6 Å, substitution of Lys-31 with alanine results in a 100-fold loss of affinity for A 18 (35,36) and likely reflects its importance in providing a positive electrostatic surface to complement the negatively charged RNA sugar phosphate backbone and in productive electrostatic steering of polynucleotides to the binding pockets of the distal face (41,42). Pyrimidines could reciprocate the donor-acceptor hydrogen bonding pattern of the peptide backbone in the A site, but would require the base to be in higher-energy syn conformation, and for uridine, require a Ϸ3 Å shift.…”

Section: Resultsmentioning

confidence: 99%

“…However, caution must be used in interpreting these latter data, as A 3 and A 6 might bind the proximal site as well as the distal site. Regardless, the significant gain in affinity of Hfq for longer A-R-N tracts can be attributed to an increase in the local concentration of these triplets, thus allowing the tract to zipper cooperatively into place, and the positive electrostatic nature of the distal face (56), which through electrostatic steering (41,42) can attract longer nucleotides more effectively. Although the distal face can accommodate 6 (A-R-N) triplets, once 5 A-R-E sites are filled, the presence of additional triplets does not affect binding affinity (compare A 16 and A 27 , the values of which parallel those reported previously) (refs.…”

Section: Hfq Binds Poly(a-r-n) and Polymentioning

confidence: 99%

Structure of Escherichia coli Hfq bound to polyriboadenylate RNA

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Valentin‐Hansen

Brennan

2009

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

325

460

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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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Kinetic analysis of operator binding by the E. coli methionine repressor highlights the role(s) of electrostatic interactions

Cited by 12 publications

References 27 publications

MetJ repressor interactions with DNA probed by in-cell NMR

MetJ repressor interactions with DNA probed by in-cell NMR

Structure of Escherichia coli Hfq bound to polyriboadenylate RNA

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

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