Searching for the Nik operon: how a ligand-responsive transcription factor hunts for its DNA binding site

Abstract

Transcription factors regulate a wide variety of genes in the cell and play a crucial role in maintaining cellular homeostasis. A major unresolved issue is how transcription factors find their specific DNA binding sequence in the vast expanse of the cell and how they do so at rates that appear faster than the diffusion limit. Here, we relate an atomic-detail model that has been developed to describe the transcription factor NikR's mechanism of DNA binding to the broader theories of how transcription factors find their binding sites on DNA. NikR is the nickel regulatory transcription factor for many bacteria, and NikR from Escherichia coli is one of the best studied ligand-mediated transcription factors. For the E. coli NikR protein, there is a wide variety of structural, biochemical, and computational studies that provide significant insight into the NikR-DNA binding mechanism. We find that the two models, the atomic-level model for E. coli NikR and the cellular model for transcription factors in general, are in agreement, and the details laid out by the NikR system may lend additional credence to the current models for transcription factors searching for DNA.

Figure 1

Crystal structures of full-length E. coli NikR. (a) Apo-NikR structure with disordered α3 helices in an “open” conformation. (b) Ni²⁺-bound NikR in an open conformation with ordered α3 helices and nickel ions represented as green spheres and an inset of the high-affinity nickel site (green spheres). (c) NikR−DNA complex with NikR in a “down-cis” conformation with potassium ions (purple spheres) bound between the MBD and RHH domains and an inset view of the potassium site. The NikR−DNA complex is shown in surface representation to illustrate contacts between NikR and DNA, and the DNA motif responsible for specific recognition by NikR is indicated.

Figure 2

Nickel binding sites of NikR. (a) NikR with nickel ions bound to high-affinity and low-affinity nickel sites. (b) NikR−DNA complex with nickel ions bound to both high-affinity and low-affinity nickel sites. Nickels in high-affinity nickel sites are shown as green spheres, and low-affinity nickel ions are colored blue, with potassium ions colored purple.

Figure 3

Potassium site that links an extended H-bonding network between NikR and DNA. The MBD is colored green, and the RHH domains are colored purple. Nonspecific DNA contacts are shown as black dashed lines; hydrogen bonds within the protein linked to the potassium site are represented by cyan dashed lines, and specific contacts between NikR and DNA are indicated between the ribbons of the RHH domains and nucleotide residues as dashed red lines. Residues involved in hydrogen bonding with DNA or the hydrogen bonding network connecting the potassium site to the DNA are shown as sticks with labels. Nitrogens are colored blue, oxygens red, phosphorus orange, and carbon atoms are colored by the region of the structure.

Figure 4

Schemes illustrating the proposed mechanism for the ligand (nickel)-induced transcription factor (NikR) binding to DNA. (a) Simple, incorrect model for depicting the mechanism described for most ligand-binding transcription factors. (b) Alternate model for NikR−DNA binding indicating the importance of the high-affinity nickel ions in ordering the central α3 helices. (c) Expansion of the model shown in panel b, which considers the role that excess nickel ions may play in enhancing NikR’s affinity for DNA by binding NikR either before or after it has bound to DNA. The tetrameric metal binding domain is represented with a gray rectangle. The dimeric ribbon−helix−helix domains are represented as triangles. The DNA is represented as a ladder. Nickel ions are represented as black circles. Potassium ions are represented as gray circles. α3 helices are represented as ovals.

Figure 5

Proposed model for NikR’s search for the nik operon on DNA. The RHH domains are represented as gray triangles. The MBD is represented as a rectangle. α3 helices are represented as ovals. Nickel ions are represented as black circles and potassium ions as gray circles. DNA is represented as a black double helix. The two DNA sites highlighted in gray represent the two half-sites just upstream of the nik operon to which NikR specifically binds in the cell to repress the transcription of nikABCDE, thus indicating the site where NikR has the highest affinity for DNA.

Figure 6

Hypothetical energy landscape for NikR when bound to DNA. In panel (a), both the orientation of the RHH domains and the position on DNA are considered. When the RHH domains are in the down-cis position and NikR is at the correct DNA binding sequence, there is an energy minimum (blue well). As the RHH domains assume a more “out” position, the energy landscape becomes more rugged with many shallow minima. (b) Cross section of the energy landscape, corresponding to the extreme orientation of the RHH domains in the nonspecific or up-cis DNA binding state of NikR. (c) Different cross section of the energy landscape corresponding to an orientation in which the RHH domains adopt the specific or down-cis DNA binding state of NikR. Four energy minima are shown for the two DNA binding modes and represent four different DNA subsequences, i.e., four different sites on the DNA polymer.