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Curr Opin Microbiol. Author manuscript; available in PMC Apr 1, 2010.
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PMCID: PMC2688824
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Allostery in the LacI/GalR Family: Variations on a Theme

Summary

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.

Overview of the LacI/GalR family

In virtually all bacteria, LacI/GalR family members regulate transcription for a wide range of processes. First catalogued in 1992 by Weickert and Adhya [1], sequences of >1000 characterized and hypothetical homologues are now known (2008 BLAST search of Swiss-Prot). These proteins have not been found in archaebacteria or eukaryotes, although proteins with homologous domains are ubiquitous.

The LacI/GalR family can be divided into >33 paralogue groups that appear to derive from an ancestral gene. As many as 22 paralogues co-exist in a single species. Many members coordinate available nutrients with expression of catabolic genes [1], but some regulate processes as diverse as nucleotide biosynthesis and toxin expression (e.g. [2,3]). Two members are “master” regulators: homologues CcpA and CRA control expression of enzymes that determine carbon flow in Gram-negative and Gram-positive bacteria, respectively. If these key proteins are disabled, virulence is altered in several pathogens (e.g. [4,5•, 6•,7]).

The common function of the LacI/GalR proteins, which features allosteric regulation of DNA binding to modulate transcription, is shown in Figure 1. Each homologue has evolved a unique variation: In addition to binding specific “operator” DNA sequences, each protein exhibits specificity for distinct effector ligands. Although most members repress transcription, some act as both repressors and activators (e.g. CcpA, as reviewed in [8•]). Some homologues control one operon (e.g., LacI), whereas others coordinate a set of related operons — for example, CRA controls >10 operons and PurR regulates at least 19 [912]). Binding the effector ligand may either decrease (induction) or increase DNA-binding affinity (co-repression), thereby altering transcription levels of downstream genes (Figure 1). As might be anticipated in a regulatory loop, effector molecules are frequently metabolically related to the regulated operon (e.g. [1,3,9,13,14]). In addition to or instead of small molecules, some family members bind other proteins [1,1517].

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 ...

The common monomeric structure of the LacI/GalR proteins comprises both DNA-binding and regulatory domains (Figure 2). Homodimer formation is required for high-affinity binding to operator DNA, which is usually some variation on an inverted repeat sequence (Figure 2, [1]). The two functional domains are linked by ~18 amino acids that mediate key interactions (see below). In the LacI/GalR family, the regulatory domains have two essential roles: (i) They receive and transmit the “input” signal from binding the effector molecule, and (ii) they mediate homodimer formation [1,8•,1820].

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 ...

Functional and allosteric variation within the LacI/GalR family

Paradigm of an inducible repressor — LacI with allolactose or IPTG

E. coli lactose repressor protein (LacI) represses the lac operon until it binds the physiological inducer allolactose or the gratuitous inducer IPTG (reviewed in [13,21]). The LacI dimer can effect repression and induction of the lac operon through binding a single high affinity operator [22,23]. In addition, wild-type LacI contains a sequence of ~20 amino acids at the C-terminus of the regulatory domain that promotes tetramer formation, allowing stronger repression through DNA-looping with two operator sequences (reviewed in [13]) (Figure 3). These loops have been visualized directly in single molecule experiments [24•]. At low in vivo inducer concentrations, one dimer within the tetramer appears to stochastically dissociate from the primary operator, leading to small bursts of gene expression [25••]. High inducer concentrations lead to LacI dissociation from both operators, increasing the duration of large bursts of gene expression [25••].

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 ...

Induced LacI remains capable of binding DNA, but the affinity for the operator site is reduced ≥3 orders of magnitude, allowing excess genomic, nonspecific DNA to compete for the repressor protein. Indeed, LacI seldom dissociates from DNA in vivo [26]. The number of inducers that elicit induction is unknown: Thermodynamic evidence is consistent with 2 inducers/dimer [27], but others argue that one is sufficient [28]. Perhaps complexes with 0, 1, and 2 inducers bound/dimer result in distinct states with different DNA-binding properties. Gratuitous anti-inducer ligands are known that enhance LacI affinity for operator DNA, whereas ”neutral” ligands bind the same effector site but elicit no change in DNA-binding affinity [27,29]

Despite extensive efforts, no high resolution structure shows a complete picture for even a single functional state of LacI (e.g. [20,3033]). Nonetheless, these structures have been invaluable for successive analyses of allostery: Comparison of the LacI·OsymDNA·anti-inducer and LacI·inducer structures led to the hypothesis that inducer binding shifts the N-subdomains of the regulatory domain [20,32]. These changes would ultimately impact the spacing of the N-terminal DNA-binding domains, misaligning the sites and lowering affinity. Motions between these two regulatory domain conformations were simulated with targeted molecular dynamics [34]. The predicted structural intermediates are in good agreement with existing experimental data and provide the basis for ongoing studies of LacI allostery (e.g., [27,35]).

The newest X-ray structures of LacI bound to either anti-inducer or neutral ligands show very few changes in the regulatory domain compared to the inducer-bound regulatory domain structure [36•]. Thus, these structures — including the LacI•IPTG structure — might represent “off-pathway” conformations. True allosteric changes might be seen only in the DNA-bound ternary complexes. To that end, small-angle X-ray scattering was carried out with full-length tetrameric and dimeric LacI bound to DNA and to DNA/IPTG [37]. Only subtle conformational changes occur within the dimer•operator complex upon IPTG binding. Notably, the linker region that is extended in apo-LacI is compact in the both LacI•DNA and the induced LacI•DNA•IPTG complex. In tetrameric LacI, inducer binding led to a change in the dimer•dimer disposition, reflecting the inherent flexibility of the tetrameric arrangement.

Paradigm of a repressible system — PurR with guanine or hypoxanthine

In E. coli, the purine repressor protein (PurR) regulates 19 operons that control purine and pyrimidine metabolic pathways (e.g. [11,12]). The physiological allosteric response of PurR is opposite to that of LacI — high affinity operator binding requires the presence of co-repressor ligand [3,38,39] (Figure 1B). Co-repressors are guanine or hypoxanthine [3]. When DNA-binding affinity is measured in the presence and absence of co-repressor, the allosteric response of PurR is about 2 orders of magnitude [39], significantly smaller than LacI induction, but near that observed for anti-inducers on LacI [40•]. As with LacI, the stoichiometry of PurR:co-repressor required to elicit the allosteric effect is unknown.

PurR crystallizes more readily than most other family members, and a number of structures are available for wild-type and mutant homodimers bound to DNA and a variety of co-repressors (e.g. [4145]). As with LacI, structures are not known for all possible functional states. Comparing structures of the apo-regulatory domain and corepressed full-length PurR, Brennan and colleagues hypothesized that large subdomain domain motions separate the DNA-binding domains too far to bind the operator half-sites [42]. The Mowbray lab [46] showed that effector binding to PurR exhibits a larger reorientation of the regulatory subdomains than does LacI. However, small-angle X-ray scattering results with a chimera comprising the LacI DNA-binding domain and the PurR regulatory domain show much smaller changes than LacI [47••]. This outcome may be an effect of either chimera formation or truncation of the PurR DNA-binding domain in the apo-PurR structure.

Paradigm of a homologue with a protein effector — CcpA

In Gram-positive bacteria, carbon catabolite protein A (CcpA) is a central regulator of carbon metabolism, controlling hundreds of genes; this homologue can function either as a repressor or an activator (reviewed in [8•,48]). Several structures of CcpA have been solved (e.g. [8•,49,50]. Unlike LacI and PurR, the primary allosteric effectors of CcpA are the proteins HPr or Crh [51]. These cofactor proteins are phosphorylated at Ser46 under particular metabolic conditions. In turn, one phosphorylated cofactor binds to each monomer within a CcpA dimer, facilitating a structural change to a “closed” form and enhancing DNA binding [51] (see Figure 1B). Interestingly, binding to different cofactor proteins can affect regulation of different operons (reviewed in [8•]). The HPr-Ser46-P/Crh-Ser46-P binding site is not the same as for the small molecule effector, but lies near residues on the three strands that link the N-and C-subdomains (Figure 2, yellow region). Upon phosphoprotein binding, the conformational change seen in the CcpA regulatory domain is similar to that seen when LacI and PurR bind small effector ligands. CcpA can also bind either glucose-6-phosphate or fructose-1,6-bisphosphate in the canonical effector binding site, which enhances the cofactor function of HPr-Ser46-P but not Crh-Ser46-P (see [8•]). Interactions with HPr-Ser46-P are also observed for the B. subtilis homologue RbsR [52].

A distinctive paradigm — CytR

The E. coli cytidine repressor protein (CytR) regulates at least nine transcriptional units encoding genes involved in purine and pyrimidine biosynthesis and utilization (reviewed in [53]). CytR binds to cytO DNA as a homodimer; DNA-binding is cooperative in the presence of two flanking catabolite repressor proteins (CRP) (Figure 4). Notably, the spacing of cytO half-sites is varied and can be much wider than for the other LacI/GalR proteins [54,55]. CytR binding to its small molecule effector (cytidine) has no effect on intrinsic DNA binding affinity (e.g. [56,57••]). Instead, the cytidine-induced conformational change disallows simultaneous CytR contacts with CRP and cytO. As a result (i) cooperative DNA binding of CytR and CRP is diminished, allowing RNA polymerase to compete for cytO, and (ii) direct interactions between CRP and RNA polymerase are altered [56, 57••, 58].

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 ...

Some of the differences in CytR function may arise from differences in the sequence linking the two functional domains (see below). No high resolution structure has been obtained, but biophysical data suggest that CytR can adopt multiple conformations in the apo-state that are constrained differently when bound to operators with distinct half-site spacings [57••]. Unlike many members of the LacI/GalR family, altered CytR•CRP interactions provide a “rheostatic” rather than “on/off” switching mechanism.

Emerging structure/function relationships in the LacI/GalR family

The regulatory domain — allostery and adaptability

The regulatory domain contains the effector and cognate protein binding sites, making this region the basic element for allostery (Figure 2). Structural changes of this domain are currently illuminated by comparison of the apo- and ligand-bound structures. LacI, PurR, and CcpA appear to have a common cleft closure, in which the N-subdomain moves and the C-subdomain remains fixed [32,42,46,50]. Changes in the regulatory domain appear to dictate the direction of allosteric response for the intact protein, as indicated by studies with chimeric repressors: When the LacI DNA-binding domain and linker are fused to the PurR regulatory domain, the chimera is co-repressed by hypoxanthine [47••], whereas when fused to the GalR regulatory domain, the chimera is induced by galactose [59•]. In LacI and GalR, several mutants that cannot respond to effector are found in the regulatory domain, in either the effector binding pocket or in regions that are crucial for allostery [60,61].

Despite its dominant role, the regulatory domain can be adapted for various functions. In addition to accommodating diverse specificities for different effectors, the regulatory domain can be either induced or co-repressed. Indeed, these alternate phenomena can occur on the same regulatory domain. As mentioned previously, LacI binds inducers, anti-inducers, and neutral ligands. Moreover, isothermal titration calorimetry experiments showed that ONPG, a neutral ligand for tetrameric LacI, behaved as an anti-inducer for dimeric LacI [40•]. The E. coli homologue GalR also has inducer (galactose and fucose) and anti-inducer (paradoxically, IPTG) ligands [62]. Although we presented PurR co-repression as “opposite” to LacI induction, a better comparison might well be the LacI•DNA•anti-inducer relationship.

Based on these observations, we propose that all LacI/GalR regulatory domains have potential for multiple allosteric modes. For example, a gratuitous inducer might be identified for PurR. Further, mutations that arise in evolution or are designed in the laboratory might influence the allosteric effect of ligand. Such latent allosteric potential in an ancestral regulatory domain would enable an inducible regulator to evolve the co-repression required to shutdown biosynthetic pathways (and vice versa).

The linker sequence — allosteric propagation

The 18 amino acids that join the DNA-binding domain to the regulatory domain are involved in many interfaces. These are best understood by subdividing the linker into an unstructured N-linker, a central hinge helix, and an unstructured C-linker (Figure 2). One face of the hinge helix directly contacts DNA; another face forms an interface between the two helices of a dimer; and other helix residues interact with the regulatory domain. In addition, both the N- and C-linkers interact with the regulatory domain. From the available structures, hypotheses have been formed about how structural changes are propagated to and through the respective linkers (see above). However, the only structural information available on the true allosteric complexes is low resolution from small angle X-ray scattering [37,47••]. These data show that the LacI linker remains compact in the DNA complexes of either full-length LacI or an engineered chimera comprising LacI and PurR (“LLhP”).

Even though linker conformational changes may be small, mutagenesis illuminates several positions important to allostery. Formation of a disulfide bond between the LacI linkers abolished allostery for some operators, whereas inserted glycines diminished the allosteric response [63,64]. Some amino acid substitutions of the LacI C-linker position 61 abolish inducibility [60]. Other substitutions at the same residue in LLhP dramatically enhanced the magnitude of the allosteric response to co-repressor [47••]. Mutagenesis of a second chimera (comprising the LacI DNA-binding domain and the GalR regulatory domain) suggests that at least four additional linker positions may participate in allostery [59•]. Because many of these substituted positions are not conserved among family members, the effects of mutagenesis might mirror the evolution of allosteric differences between family members.

Many family members posses a conserved linker motif: Y/FxPxxxAxxL/M. A key feature is the alternative L/M side chain, which inserts into the minor groove in the center of the DNA operator [20,42,50]. A few family members lack features of the motif and/or have multiple P or G residues that are anticipated to disrupt the hinge helix. In the bacterial phylum Firmicutes, homologues that lack the linker motif also have a distinct operator motif [65]. Thus, the larger LacI/GalR family can be divided into two subfamilies [59•,65], which appear to have evolved different mechanisms by which the linkers bind DNA and convey allostery. For example, E. coli CytR lacks the L/M, has a P and a G in the “helical” region, and cytO is similar to the operator subfamily identified in Firmicutes. The linker of CytR appears to adopt multiple conformations, allowing this repressor to recognize variable spacing and rotations in the cytO half-sites (Figure 4) [57••].

DNA as an allosteric effector of LacI/GalR proteins

Thermodynamically, allostery occurs when binding to ligand A differs in the absence and presence of ligand B. To preserve a complete thermodynamic cycle, the complement must also occur. Since effector binding to LacI/GalR proteins alters DNA binding affinity, DNA binding by the LacI/GalR proteins must alter effector binding, a feature that has been directly measured (e.g., [39]). Given this behavior, each specific operator sequence might exhibit a different allosteric response to small molecule effectors. This relationship has been confirmed for variants of LacI [63,64] and chimera LLhP [47••], and conceivably could contribute to the operator-specific responses seen with variants of CcpA [66•]. Many LacI/GalR proteins are known to regulate multiple operons, and an alternative allosteric response to various DNA sequences would allow their differential, but simultaneous, regulation.

Concluding remarks

The ubiquity of LacI/GalR regulatory proteins in prokaryotes testifies to the robust nature of this mechanism for conserving the energy required for mRNA and protein production [67•]. Their conserved structure has potential to be regulated by small molecules, by other proteins, or their combination. The protein structure is adaptable, demonstrating both induction and co-repression within the same molecule. The structure can effect on/off switching — with >1,000-fold change in transcription — or can rheostatically modulate gene expression between ~10 and 100-fold. As we understand the intricacies of the LacI/GalR proteins, and the ways in which they can be varied, we gain the capacity to introduce “designed” regulatory systems into the cellular milieu.

Acknowledgments

We are grateful for the support of the National Institutes of Health (GM079423 and P20 RR17708 for LSK, GM22441 for KSM) and the Robert A. Welch Foundation (C-576 for KSM).

Footnotes

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References and recommended reading

Papers of particular interest, published within the period of the review have been highlighted as:

• of special interest

•• of outstanding interest

1. Weickert MJ, Adhya S. A family of bacterial regulators homologous to Gal and Lac repressors. J Biol Chem. 1992;267:15869–15874. [PubMed]
2. Colmer JA, Hamood AN. Characterization of ptxS, a Pseudomonas aeruginosa gene which interferes with the effect of the exotoxin A positive regulatory gene, ptxR. Mol Gen Genet. 1998;258:250–259. [PubMed]
3. Meng LM, Nygaard P. Identification of hypoxanthine and guanine as the co-repressors for the purine regulon genes of Escherichia coli. Mol Microbiol. 1990;4:2187–2192. [PubMed]
4. Seidl K, Stucki M, Ruegg M, Goerke C, Wolz C, Harris L, Berger-Bächi B, Bischoff M. Staphylococcus aureus CcpA affects virulence determinant production and antibiotic resistance. Antimicrob Agents Chemother. 2006;50:1183–1194. [PMC free article] [PubMed]
5• . Shelburne SA, III, Keith D, Horstmann N, Sumby P, Davenport MT, Graviss EA, Brennan RG, Musser JM. A direct link between carbohydrate utilization and virulence in the major human pathogen group A Streptococcus. Proc Natl Acad Sci USA. 2008;105:1698–1703. In group A Streptococcus, transcription of several virulence factors is related to carbohydrate utilization. Bacterial variants with a deleted CcpA gene show decreased virulence in a mouse model of invasive infection and changed expression of virulence genes on a nutrient-limited growth media. Further, purified CcpA bound directly to the promoter region of the virulence gene for steptolysin S. [PMC free article] [PubMed]
6•. Kinkel TL, McIver KS. CcpA-mediated repression of streptolysin S expression and virulence. Infect Immun. 2008;76:3451–3463. A CcpA deletion mutant of a group A Steptococcus strain shows increased virulence in a mouse model. See Shelburne et al. [5] which reported diminished virulence in a very similar strain. The apparent paradox between these two studies remains unresolved, but both studies show a definitive link between CcpA function and virulence. This manuscript provides citations for studies of other pathogenic bacteria in which virulence has been linked to CcpA function. [PMC free article] [PubMed]
7. Allen JH, Utley M, van Den Bosch H, Nuijten P, Witvliet M, McCormick BA, Krogfelt KA, Licht TR, Brown D, Mauel M, et al. A functional cra gene is required for Salmonella enterica serovar typhimurium virulence in BALB/c mice. Infect Immun. 2000;68:3772–3775. [PMC free article] [PubMed]
8•. Schumacher MA, Seidel G, Hillen W, Brennan RG. Structural mechanism for the fine tuning of CcpA function by the small molecule effectors glucose 6-phosphate and fructose 1,6-bisphosphate. J Mol Biol. 2007;368:1042–1050. Previous structural studies by this lab demonstrated that phosphoprotein HPr binds to CcpA outside of the canonical effector binding site of the regulatory domain. Here, the structural means by which glucose-6-phosphate and fructose-1,6-diphosphate further enhance DNA binding is shown. The small molecules bind in the effector site and appear to “buttress” the DNA-binding conformation, bolstering interactions between CcpA and phosphorylated HPr. [PubMed]
9. Saier MH., Jr Cyclic AMP-independent catabolite repression in bacteria. FEMS Microbiol Lett. 1996;138:97–103. [PubMed]
10. Shimada T, Fujita N, Maeda M, Ishihama A. Systematic search for the Cra-binding promoters using genomic SELEX system. Genes Cells. 2005;10:907–918. [PubMed]
11. Choi KY, Lu F, Zalkin H. Mutagenesis of amino acid residues required for binding of corepressors to the purine repressor. J Biol Chem. 1994;269:24066–24072. [PubMed]
12. Mironov AA, Koonin EV, Roytberg MA, Gelfand MS. Computer analysis of transcription regulatory patterns in completely sequenced bacterial genomes. Nucl Acids Res. 1999;27:2981–2989. [PMC free article] [PubMed]
13. Matthews KS, Nichols JC. Lactose repressor protein: Functional properties and structure. Prog Nucleic Acid Res Mol Biol. 1998;58:127–164. [PubMed]
14. Weickert MJ, Adhya S. The galactose regulon of Escherichia coli. Mol Microbiol. 1993;10:245–251. [PubMed]
15. Choy HE, Park SW, Aki T, Parrack P, Fujita N, Ishihama A, Adhya S. Repression and activation of transcription by Gal and Lac repressors: Involvement of alpha subunit of RNA polymerase. EMBO J. 1995;14:4523–4529. [PMC free article] [PubMed]
16. Fujita Y, Miwa Y, Galinier A, Deutscher J. Specific recognition of the Bacillus subtilis gnt cis-acting catabolite-responsive element by a protein complex formed between CcpA and seryl-phosporylated HPr. Mol Microbiol. 1995;17:953–960. [PubMed]
17. Meibom KL, Kallipolitis BH, Ebright RH, Valentin-Hansen P. Identification of the subunit of cAMP receptor protein (CRP) that functionally interacts with CytR in CRP-CytR-mediated transcriptional repression. J Biol Chem. 2000;275:11951–11956. [PubMed]
18. Choi KY, Zalkin H. Structural characterization and corepressor binding of the Escherichia coli purine repressor. J Bacteriol. 1992;174:6207–6214. [PMC free article] [PubMed]
19. Kristensen HH, Valentin-Hansen P, Søgaard-Andersen L. CytR/cAMP-CRP nucleoprotein formation in E. coli: The CytR repressor binds its operator as a stable dimer in a ternary complex with cAMP-CRP. J Mol Biol. 1996;260:113–119. [PubMed]
20. Bell CE, Lewis M. A closer view of the conformation of the Lac repressor bound to operator. Nat Struct Biol. 2000;7:209–214. [PubMed]
21. Wilson CJ, Zhan H, Swint-Kruse L, Matthews KS. The lactose repressor system: Paradigms for regulation, allosteric behavior and protein folding. Cell Mol Life Sci. 2007;64:3–16. [PubMed]
22. Oehler S, Eismann ER, Krämer H, Müller-Hill B. The three operators of the lac operon cooperate in repression. EMBO J. 1990;9:973–979. [PMC free article] [PubMed]
23. Chen J, Matthews KS. Deletion of lactose repressor carboxyl-terminal domain affects tetramer formation. J Biol Chem. 1992;267:13843–13850. [PubMed]
24•. Wong OK, Guthold M, Erie DA, Gelles J. Interconvertible Lac repressor-DNA loops revealed by single-molecule experiments. PLoS Biol. 2008;6:e232. Using single-molecule structural and kinetic methods, Wong et al. visualize small stable loops that form between the LacI tetramer and two operators. The loops form rapidly, even when operator spacings are not aligned on the same face of the DNA. Transitions between two distinct looped structures suggest a dynamic equilibrium of the tetrameric repressor protein between conformations that have different juxtapositions of its component dimers. [PMC free article] [PubMed]
25••. Choi PJ, Cai L, Frieda K, Xie XS. A stochastic single-molecule event triggers phenotype switching of a bacterial cell. Science. 2008;322:442–446. A fluorescently labeled product of the lac operon was used to visualize gene expression within single cells. At intermediate inducer concentrations, two phenotypes — induced and uninduced — are observed in a genetically identical cell population. This phenomenon is hypothesized to arise from stochastic dissociation of LacI from one operator in a looped complex, presumably the primary operator near the transcription start site. At high inducer concentrations, complete dissociation from both operators results in full induction of the operon. [PMC free article] [PubMed]
26. Elf J, Li GW, Xie XS. Probing transcription factor dynamics at the single-molecule level in a living cell. Science. 2007;316:1191–1194. [PMC free article] [PubMed]
27. Swint-Kruse L, Zhan H, Matthews KS. Integrated insights from simulation, experiment, and mutational analysis yield new details of LacI function. Biochemistry. 2005;44:11201–11213. [PubMed]
28. Oehler S, Alberti S, Müller-Hill B. Induction of the lac promoter in the absence of DNA loops and the stoichiometry of induction. Nucl Acids Res. 2006;34:606–612. [PMC free article] [PubMed]
29. Barkley MD, Riggs AD, Jobe A, Burgeois S. Interaction of effecting ligands with lac repressor and repressor-operator complex. Biochemistry. 1975;14:1700–1712. [PubMed]
30. Friedman AM, Fischmann TO, Steitz TA. Crystal structure of lac repressor core tetramer and its implications for DNA looping. Science. 1995;268:1721–1727. [PubMed]
31. Slijper M, Bonvin AM, Boelens R, Kaptein R. Refined structure of lac repressor headpiece (1–56) determined by relaxation matrix calculations from 2D and 3D NOE data: Change of tertiary structure upon binding to the lac operator. J Mol Biol. 1996;259:761–773. [PubMed]
32. Lewis M, Chang G, Horton NC, Kercher MA, Pace HC, Schumacher MA, Brennan RG, Lu P. Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Science. 1996;271:1247–1254. [PubMed]
33. Kalodimos CG, Biris N, Bonvin AM, Levandoski MM, Guennuegues M, Boelens R, Kaptein R. Structure and flexibility adaptation in nonspecific and specific protein-DNA complexes. Science. 2004;305:386–389. [PubMed]
34. Flynn TC, Swint-Kruse L, Kong Y, Booth C, Matthews KS, Ma J. Allosteric transition pathways in the lactose repressor protein core domains: Asymmetric motions in a homodimer. Protein Sci. 2003;12:2523–2541. [PMC free article] [PubMed]
35. Zhan H, Sun Z, Matthews KS. Functional impact of polar and acidic substitutions in the lactose repressor hydrophobic monomer•monomer interface with a buried lysine. Biochemistry. 2008 in press. [PubMed]
36•. Daber R, Stayrook S, Rosenberg A, Lewis M. Structural analysis of Lac repressor bound to allosteric effectors. J Mol Biol. 2007;370:609–619. Inducer, anti-inducer, and neutral compounds bind to LacI in the same canonical effector binding site, forming hydrogen bonds between the protein and sugar hydroxyl groups. The O6 hydroxyl of the galactoside appears key to forming a water-mediated hydrogen-bond network that engages both subdomains of the regulatory domain. The authors postulate that alterations in the hydrogen bonding network are central to the allosteric transitions of LacI. [PMC free article] [PubMed]
37. Taraban M, Zhan H, Whitten AE, Langley DB, Matthews KS, Swint-Kruse L, Trewhella J. Ligand-induced conformational changes and conformational dynamics in the solution structure of the lactose repressor protein. J Mol Biol. 2008;376:466–481. [PMC free article] [PubMed]
38. Rolfes RJ, Zalkin H. Purification of the Escherichia coli purine regulon repressor and identification of corepressors. J Bacteriol. 1990;172:5637–5642. [PMC free article] [PubMed]
39. Moraitis MI, Xu H, Matthews KS. Ion concentration and temperature dependence of DNA binding: Comparison of PurR and LacI repressor proteins. Biochemistry. 2001;40:8109–8117. [PubMed]
40• . Wilson CJ, Zhan H, Swint-Kruse L, Matthews KS. Ligand interactions with lactose repressor protein and the repressor-operator complex: The effects of ionization and oligomerization on binding. Biophys Chem. 2007;126:94–105. Isothermal titration calorimetry shows that LacI binding to inducer IPTG is driven by enthalpic forces, whereas a weaker inducer has low enthalpic contributions. Further, changes in pH and/or oligomerization (dimer vs tetramer) convert a neutral ligand into an anti-inducer. A wide range of energetic consequences occur when LacI binds to structurally similar ligands, indicating the sensitivity of binding processes and the range of effects that can occur. [PubMed]
41. Nagadoi A, Morikawa S, Nakamura H, Enari M, Kobayashi K, Yamamoto H, Sampei G, Mizobuchi K, Schumacher MA, Brennan RG, et al. Structural comparison of the free and DNA-bound forms of the purine repressor DNA-binding domain. Structure. 1995;3:1217–1224. [PubMed]
42. Schumacher MA, Choi KY, Lu F, Zalkin H, Brennan RG. Mechanism of corepressor-mediated specific DNA binding by the purine repressor. Cell. 1995;83:147–155. [PubMed]
43. Schumacher MA, Glasfeld A, Zalkin H, Brennan RG. The X-ray structure of the PurR-guanine-purF operator complex reveals the contributions of complementary electrostatic surfaces and a water-mediated hydrogen bond to corepressor specificity and binding affinity. J Biol Chem. 1997;272:22648–22653. [PubMed]
44. Glasfeld A, Koehler AN, Schumacher MA, Brennan RG. The role of lysine 55 in determining the specificity of the purine repressor for its operators through minor groove interactions. J Mol Biol. 1999;291:347–361. [PubMed]
45. Huffman JL, Lu F, Zalkin H, Brennan RG. Role of residue 147 in the gene regulatory function of the Escherichia coli purine repressor. Biochemistry. 2002;41:511–520. [PubMed]
46. Mowbray SL, Björkman AJ. Conformational changes of ribose-binding protein and two related repressors are tailored to fit the functional need. J Mol Biol. 1999;294:487–499. [PubMed]
47••. Zhan H, Taraban M, Trewhella J, Swint-Kruse L. Subdividing repressor function: DNA binding affinity, selectivity, and allostery can be altered by amino acid substitution of nonconserved residues in a LacI/GalR homologue. Biochemistry. 2008;47:8058–8069. The LacI DNA-binding domain and linker were fused to the PurR regulatory domain. The chimeric protein, LLhP, does not discriminate between DNA sequences as well as LacI, and the linker structure is significantly more compact in the absence of DNA. Additional amino acid changes in the LLhP linker result in a range of functional effects. Thus, variation of linker residues can modulate different aspects of repressor function. Two point mutations increased the LLhP allosteric response by an order of magnitude. [PMC free article] [PubMed]
48. Sonenshein AL. Control of key metabolic intersections in Bacillus subtilis. Nat Rev Microbiol. 2007;5:917–927. [PubMed]
49. Loll B, Saenger W, Biesiadka J. Structure of full-length transcription regulator CcpA in the apo form. Biochim Biophys Acta. 2007;1774:732–736. [PubMed]
50. Schumacher MA, Allen GS, Diel M, Seidel G, Hillen W, Brennan RG. Structural basis for allosteric control of the transcription regulator CcpA by the phosphoprotein HPr-Ser46-P. Cell. 2004;118:731–741. [PubMed]
51. Galinier A, Deutscher J, Martin-Verstraete I. Phosphorylation of either Crh or HPr mediates binding of CcpA to the Bacillus subtilis xyn cre and catabolite repression of the xyn operon. J Mol Biol. 1999;286:307–314. [PubMed]
52. Müller W, Horstmann N, Hillen W, Sticht H. The transcription regulator RbsR represents a novel interaction partner of the phosphoprotein HPr-Ser46-P in Bacillus subtilis. FEBS J. 2006;273:1251–1261. [PubMed]
53. Senear DF, Perini LT, Gavigan SA. Analysis of interactions between CytR and CRP at CytR-regulated promoters. Methods Enzymol. 1998;295:403–424. [PubMed]
54. Pedersen H, Valentin-Hansen P. Protein-induced fit: The CRP activator protein changes sequence-specific DNA recognition by the CytR repressor, a highly flexible LacI member. EMBO J. 1997;16:2108–2118. [PMC free article] [PubMed]
55. Jørgensen CI, Kallipolitis BH, Valentin-Hansen P. DNA-binding characteristics of the Escherichia coli CytR regulator: A relaxed spacing requirement between operator half-sites is provided by a flexible, unstructured interdomain linker. Mol Microbiol. 1998;27:41–50. [PubMed]
56. Pedersen H, Søgaard-Andersen L, Holst B, Valentin-Hansen P. Heterologous cooperativity in Escherichia coli. The CytR repressor both contacts DNA and the cAMP receptor protein when binding to the deoP2 promoter . J Biol Chem. 1991;266:17804–17808. [PubMed]
57••. Tretyachenko-Ladokhina V, Cocco MJ, Senear DF. Flexibility and adaptability in binding of E. coli cytidine repressor to different operators suggests a role in differential gene regulation. J Mol Biol. 2006;362:271–286. Thermodynamic analyses of CytR binding to natural and synthetic operators demonstrate effects that correlate with the spacing between the two operator half-sites. These effects are mediated by the dynamic structure of the dimeric protein, with different limitations imposed by binding to variant operator DNA sequences. In this fashion, DNA sequence-specific effects are exerted on repression and induction of CytR. [PubMed]
58. Kallipolitis BH, Nørregaard-Madsen M, Valentin-Hansen P. Protein-protein communication: Structural model of the repression complex formed by CytR and the global regulator CRP. Cell. 1997;89:1101–1109. [PubMed]
59•. Meinhardt S, Swint-Kruse L. Experimental identification of specificity determinants in the domain linker of a LacI/GalR protein: Bioinformatics-based predictions generate true positives and false negatives. Proteins. 2008;73:941–957. The LacI/GalR family is frequently used in the development of bioinformatics algorithms to predict which conserved or nonconserved residues are functionally important. However, different algorithms predict different residues. Here, predictions for the linker sequence were experimentally tested using an engineered homologue. Amino acid substitutions of 6 predicted and 3 non-predicted residues show that all contribute to repressor function. Several linker sites are implicated in allostery. [PMC free article] [PubMed]
60. Suckow J, Markiewicz P, Kleina LG, Miller J, Kisters-Woike B, Müller-Hill B. Genetic studies of the Lac repressor. XV: 4000 single amino acid substitutions and analysis of the resulting phenotypes on the basis of the protein structure. J Mol Biol. 1996;261:509–523. [PubMed]
61. Zhou YN, Chatterjee S, Roy S, Adhya S. The non-inducible nature of super-repressors of the gal operon in Escherichia coli. J Mol Biol. 1995;253:414–425. [PubMed]
62. Buttin G. Regulatory mechanisms in the biosynthesis of the enzymes of galactose metabolism in Escherichia coli K 12. I. The induced biosynthesis of galactokinase and the simultaneous induction of the enzymatic sequence. [in French] J Mol Biol. 1963;7:164–182. [PubMed]
63. Falcon CM, Matthews KS. Engineered disulfide linking the hinge regions within lactose repressor dimer increases operator affinity, decreases sequence selectivity, and alters allostery. Biochemistry. 2001;40:15650–15659. [PubMed]
64. Falcon CM, Matthews KS. Operator DNA sequence variation enhances high affinity binding by hinge helix mutants of lactose repressor protein. Biochemistry. 2000;39:11074–11083. [PubMed]
65. Francke C, Kerkhoven R, Wels M, Siezen RJ. A generic approach to identify Transcription Factor-specific operator motifs; Inferences for LacI-family mediated regulation in Lactobacillus plantarum WCFS1. BMC Genomics. 2008;9:145. [PMC free article] [PubMed]
66•. Sprehe M, Seidel G, Diel M, Hillen W. CcpA mutants with differential activities in Bacillus subtilis. J Mol Microbiol Biotechnol. 2007;12:96–105. When CcpA is mutated at residues that have conformations sensitive to HPrSerP binding, the regulator retains activation of the ackA operon but loses regulation for other operons. In contrast, CcpA variants with mutations in the canonical binding site for small molecules selectively retain regulation for a repressed operon — xynP. Thus, CcpA activation and repression may involve different allosteric states. [PubMed]
67•. Stoebel DM, Dean AM, Dykhuizen DE. The cost of expression of Escherichia coli lac operon proteins is in the process, not the products. Genetics. 2008;178:1653–1660. The cost for regulating gene expression is associated with transcription and translation of the associated genes, and not with the subsequent activities of the translated proteins. The expression of a particular protein can be costly or beneficial depending on the environment. The results point to a single selective pressure for all regulation within E. coli. [PMC free article] [PubMed]
68••. Semsey S, Virnik K, Adhya S. Three-stage regulation of the amphibolic gal operon: From repressosome to GalR-free DNA. J Mol Biol. 2006;358:355–363. GalR binds to two operator DNA sites, creating a looped structure that is stabilized by binding of the HU protein and DNA supercoiling. Galactose binding to GalR promotes sequential disruption of the complex and generates different regulatory states for the gal operon. [PubMed]
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