MetJ repressor interactions with DNA probed by in-cell NMR

Abstract

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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