Allostery in the LacI/GalR family: variations on a theme

Abstract

The lactose repressor protein (LacI) was among the very first genetic regulatory proteins discovered, and more than 1000 members of the bacterial LacI/GalR family are now identified. LacI has been the prototype for understanding how transcription is controlled using small metabolites to modulate protein association with specific DNA sites. This understanding has been greatly expanded by the study of other LacI/GalR homologues. A general picture emerges in which the conserved fold provides a scaffold for multiple types of interactions - including oligomerization, small molecule binding, and protein-protein binding - that in turn influence target DNA binding and thereby regulate mRNA production. Although many different functions have evolved from this basic scaffold, each homologue retains functional flexibility: For the same protein, different small molecules can have disparate impact on DNA binding and hence transcriptional outcome. In turn, binding to alternative DNA sequences may impact the degree of allosteric response. Thus, this family exhibits a symphony of variations by which transcriptional control is achieved.

Fig. 1

Summary of LacI/GalR protein cycles for inducible and repressible systems. For inducible systems (upper panel), the oligomer binds to its target operator DNA, inhibiting RNA polymerase transcription in the absence of a small molecule effector. This ligand (red) is often a substrate for downstream genes or a metabolic product related to those genes (e.g., allolactose for LacI, galactose for GalR). In the presence of the ligand, a conformational change in the protein diminishes operator DNA binding affinity, releasing the promoter for production of mRNA. The cycle continues when effector concentration is decreased. For repressible systems (lower panel), the protein oligomer exhibits lower affinity for its target operator DNA, and transcription is unimpeded. The presence of its cognate co-repressor ligand (teal) and/or cofactor protein (gray) elicits a conformational shift in the repressor to a form with higher affinity for its target DNA site, which in turn decreases transcription. The regulated genes for repressible systems are often biosynthetic, and extremes of DNA binding (and hence expression of downstream genes) are lower than for inducible systems.

Fig. 2

Common features of LacI/GalR proteins. The structure depicts that of the PurR dimer (gray ribbons) in a complex with DNA (gold wireframe at the top of the figure) and corepressor (brown spacefilling atoms) (pdb 1wet, [43]). LacI/GalR monomers contain common structural features that include a DNA binding domain with a helix-turn-helix motif, a linker between the two major domains, and a regulatory domain that encompasses regions for oligomerization and for effector binding. The small molecule effector and known cofactor protein sites are highlighted. The fold of the DNA-binding and regulatory domains are highly conserved among the family. The linkers of LacI, PurR, and CcpA contain a hinge helix and two unstructured segments. The linker makes multiple contacts, which are outlined in the text. Two DNA binding domains of a dimer are required to bind the inverted repeat sequences of the operator DNA binding sites (right panel). These operator sequences can be direct inverted repeats or contain one or two base pairs inserted between the repeats. In a few cases (e.g., CytR [57••]), more widely spaced inverted repeats separate the sites.

Fig. 3

DNA looping by LacI/GalR proteins. Formation of DNA loops significantly enhances repression. Two types of loops can occur: (i) Proteins, such as LacI, that are tetrameric (monomers are depicted as purple circles) can bind DNA at two operator sites (one per dimer) to generate highly stable loops. (ii) Dimeric proteins, such as GalR, can bind to two different operator sites, with looping between these sites mediated by protein•protein interactions that can be promoted by DNA bending proteins (e.g., GalR and HU binding, [68••]) and/or by DNA supercoiling. Allosteric response of looped complexes differs at intermediate and high effector concentrations [25••,68••]. The relative positions of the promoters, operators, and downstream genes differ for various operons.

Fig. 4

CytR regulation. CytR is a unique variation on the LacI/GalR structural theme in that the CytR-cytO interaction is not modulated by small ligand binding (figure adapted from reference [57••]). Instead, the CytR dimer (depicted with 2 green domains per monomer) cooperatively binds DNA via interactions with the catabolite repressor protein (CRP; dimer of blue ovals). In the presence of cytidine, the CytR allosteric change appears to disallow the simultaneous contact of both cytO and CRP. As a result, RNA polymerase can compete for the cytO binding site and RNAP-CRP direct interactions are altered, allowing transcription to occur. Variations in CytR DNA-binding affinity are achieved by varying the distance between cytO half-sites. CytR has been postulated to access a range of conformations that contribute to differential regulation of variant operators [57••].