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.

Fig. 1.

Schematic of the E. coli chromosome (4,639,675 bp) showing the locations of the genes regulated by MetJ (not to scale). The gray boxes indicate the number of 8-bp metboxes at each locus, ranging from 2 to 5.

Fig. 2.

HSQC spectra of intact, living cells does not show the overexpressed MetJ spectrum. Cells overexpressing MetJ (A) show the same pattern as cells without the MetJ plasmid (B) indicating that the spectrum from MetJ is not being detected. The amide region for proton resonances is on the x axis, and amide nitrogen resonances are on the y axis. Icons represent cells with or without the Cα-backbone structure of a MetJ dimer (PDB entry 1CMB).

Fig. 3.

The MetJ spectrum can be restored in preparations exhibiting nonspecific DNA binding by DNase treatment. (A and B) The spectrum of purified MetJ dimer (A) is lost after the addition of nonspecific DNA (B). (C) The spectrum is restored with the addition of DNase. Icons represent a MetJ dimer in solution: alone, interacting with nonspecific double-stranded DNA, or with that DNA digested into fragments.

Fig. 4.

MetJ can be rescued from nonspecific DNA binding (seen in Fig. 3B) by metbox DNA. Addition of a 20-bp DNA fragment containing 2 adjacent 8-bp metbox sequences restores the HSQC fingerprint of MetJ bound to its recognition site (A) as shown by comparison with the purified complex (B). Icons represent 2 MetJ dimers interacting specifically with metbox DNA, in the presence or absence of nonspecific DNA.

Fig. 5.

The MetJ spectrum can be restored in preparations exhibiting interactions within cell lysates by DNase treatment. (A) The spectrum of purified MetJ dimer (seen in Fig. 3A) is lost after the addition of an unlabeled E. coli cell lysate. (B) The spectrum is restored by the addition of DNase. Icons represent the MetJ dimer in cell lysates, with nonspecific DNA either intact or digested.