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

Augustus, Anne Marie; Reardon, Patrick N.; Heller, William T.; Spicer, Leonard D.

doi:10.1074/jbc.m605763200

Cited by 20 publications

(18 citation statements)

References 25 publications

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“…Purified MetJ was obtained by published procedures (38). Metbox DNA was purchased from IDT as a 20-bp oligo containing 2 copies of MetJ's preferred binding site flanked by GG and CC bases: 5Ј GG-AGACGTCT-AGACGTCT-CC 3Ј.…”

Section: Methodsmentioning

confidence: 99%

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: Methodsmentioning

confidence: 99%

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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“…MetJ forms a homodimer in its native state [22]. In case of multiple adjacent metboxes it may form complexes of several homodimers arranged in a wheel-like structure around the DNA [23]. DNA binding of MetJ is regulated by its co-repressor, S-adenosylmethionine (SAM), an end product of methionine biosynthesis, Fig.…”

Section: Introductionmentioning

confidence: 99%

Dynamic Allostery in the Methionine Repressor Revealed by Force Distribution Analysis

2009

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Many fundamental cellular processes such as gene expression are tightly regulated by protein allostery. Allosteric signal propagation from the regulatory to the active site requires long-range communication, the molecular mechanism of which remains a matter of debate. A classical example for long-range allostery is the activation of the methionine repressor MetJ, a transcription factor. Binding of its co-repressor SAM increases its affinity for DNA several-fold, but has no visible conformational effect on its DNA binding interface. Our molecular dynamics simulations indicate correlated domain motions within MetJ, and quenching of these dynamics upon SAM binding entropically favors DNA binding. From monitoring conformational fluctuations alone, it is not obvious how the presence of SAM is communicated through the largely rigid core of MetJ and how SAM thereby is able to regulate MetJ dynamics. We here directly monitored the propagation of internal forces through the MetJ structure, instead of relying on conformational changes as conventionally done. Our force distribution analysis successfully revealed the molecular network for strain propagation, which connects collective domain motions through the protein core. Parts of the network are directly affected by SAM binding, giving rise to the observed quenching of fluctuations. Our results are in good agreement with experimental data. The force distribution analysis suggests itself as a valuable tool to gain insight into the molecular function of a whole class of allosteric proteins.

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“…Mutant protein (MetJ-A1C) was expressed and purified using published protocols for wildtype MetJ (15). MetJ-A1C was labeled with maleimiderhodamine (Sigma Chemicals) following manufacturer's instructions to generate MetJ-R.…”

Section: Methodsmentioning

confidence: 99%

Binding of MetJ Repressor to Specific and Nonspecific DNA and Effect of S-Adenosylmethionine on These Interactions

Augustus¹,

Sage²,

Spicer³

2010

Biochemistry

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We have used analytical ultracentrifugation to characterize the binding of the methionine repressor protein, MetJ, to synthetic oligonucleotides containing 0 to 5 specific recognition sites, called metboxes. For all lengths of DNA studied, MetJ binds more tightly to repeats of the consensus sequence than to naturally occurring metboxes, which exhibit a variable number of deviations from the consensus. Strong cooperative binding occurs only in the presence of two or more tandem metboxes, which facilitate protein-protein contacts between adjacent MetJ dimers, but weak affinity is detected even with DNA containing 0 or 1 metbox. The affinity of MetJ for all the DNA sequences studied is enhanced by the addition of SAM, the known co-factor for MetJ in the cell. This effect extends to oligos containing 0 or 1 metbox, both of which bind two MetJ dimers. In the presence of a large excess concentration of metbox DNA, the effect of cooperativity is to favor populations of DNA oligos bound by 2 or more MetJ dimers rather than a stochastic redistribution of the repressor onto all available metboxes. These results illustrate the dynamic range of binding affinity and repressor assembly that MetJ can exhibit with DNA, and the effect of the co-repressor SAM on binding to both specific and non-specific DNA.

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

Cited by 20 publications

References 25 publications

MetJ repressor interactions with DNA probed by in-cell NMR

MetJ repressor interactions with DNA probed by in-cell NMR

Dynamic Allostery in the Methionine Repressor Revealed by Force Distribution Analysis

Binding of MetJ Repressor to Specific and Nonspecific DNA and Effect of S-Adenosylmethionine on These Interactions

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