Kathleen S. Matthews

The lactose repressor system: paradigms for regulation, allosteric behavior and protein folding

Abstract.In 1961, Jacob and Monod proposed the operon model for gene regulation based on metabolism of lactose in Escherichia coli [1]. This proposal was followed by an explication of allosteric behavior by Monod and colleagues [2]. The operon model rationally depicted how genetic mechanisms can control metabolic events in response to environmental stimuli via coordinated transcription of a set of genes with related function (e.g. metabolism of lactose). The allosteric response found in the lactose repressor and many other proteins has been extended to a variety of cellular signaling pathways in all organisms. These two models have shaped our view of modern molecular biology and captivated the attention of a surprisingly broad range of scientists. More recently, the lactose repressor monomer was used as a model system for experimental and theoretical explorations of protein folding mechanisms. Thus, the lac system continues to advance our molecular understanding of genetic control and the relationship between sequence, structure and function.

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

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.

LACTOSE REPRESSOR PROTEIN : FUNCTIONAL PROPERTIES AND STRUCTURE

The lactose repressor protein (LacI), the prototype for genetic regulatory proteins, controls expression of lactose metabolic genes by binding to its cognate operator sequences in E. coli DNA. Inducer binding elicits a conformational change that diminishes affinity for operator sequences with no effect on nonspecific binding. The release of operator is followed by synthesis of mRNA encoding the enzymes for lactose utilization. Genetic, chemical and physical studies provided detailed insight into the function of this protein prior to the recent completion of X-ray crystallographic structures. The structural information can now be correlated with the phenotypic data for numerous mutants. These structures also provide the opportunity for physical and chemical studies on mutants designed to examine various aspects of lac repressor structure and function. In addition to providing insight into protein structure-function correlations, LacI has been utilized in a wide variety of applications both in prokaryotic gene expression and in eukaryotic gene regulation and studies of mutagenesis.

Multiple Intrinsically Disordered Sequences Alter DNA Binding by the Homeodomain of the Drosophila Hox Protein Ultrabithorax

During animal development, distinct tissues, organs, and appendages are specified through differential gene transcription by Hox transcription factors. However, the conserved Hox homeodomains bind DNA with high affinity yet low specificity. We have therefore explored the structure of the Drosophila melanogaster Hox protein Ultrabithorax and the impact of its nonhomeodomain regions on DNA binding properties. Computational and experimental approaches identified several conserved, intrinsically disordered regions outside the homeodomain of Ultrabithorax that impact DNA binding by the homeodomain. Full-length Ultrabithorax bound to target DNA 2.5-fold weaker than its isolated homeodomain. Using N-terminal and C-terminal deletion mutants, we demonstrate that the YPWM region and the disordered microexons (termed the I1 region) inhibit DNA binding ∼2-fold, whereas the disordered I2 region inhibits homeodomain-DNA interaction a further ∼40-fold. Binding is restored almost to homeodomain affinity by the mostly disordered N-terminal 174 amino acids (R region) in a length-dependent manner. Both the I2 and R regions contain portions of the activation domain, functionally linking DNA binding and transcription regulation. Given that (i) the I1 region and a portion of the R region alter homeodomain-DNA binding as a function of pH and (ii) an internal deletion within I1 increases Ultrabithorax-DNA affinity, I1 must directly impact homeodomain-DNA interaction energetics. However, I2 appears to indirectly affect DNA binding in a manner countered by the N terminus. The amino acid sequences of I2 and much of the I1 and R regions vary significantly among Ultrabithorax orthologues, potentially diversifying Hox-DNA interactions.

Engineered temperature compensation in a synthetic genetic clock

Significance Synthetic gene circuits are often fragile, as perturbations to cellular conditions frequently alter their behavior. This lack of robustness limits the utility of engineered gene circuits and hinders advances in synthetic biology. Here, we demonstrate that environmental sensitivity can be reduced by simultaneously engineering circuits at the protein and network levels. Specifically, we designed and constructed a synthetic genetic clock that exhibits temperature compensation—the clock’s period does not depend on temperature. This feature is nontrivial since biochemical reactions speed up with increasing temperature. To accomplish this goal, we engineered thermal-inducibility into the clock’s regulatory structure. Computational modeling predicted and experiments confirmed that this thermal-inducibility results in a clock with a stable period across a large range of temperatures. Synthetic biology promises to revolutionize biotechnology by providing the means to reengineer and reprogram cellular regulatory mechanisms. However, synthetic gene circuits are often unreliable, as changes to environmental conditions can fundamentally alter a circuit’s behavior. One way to improve robustness is to use intrinsic properties of transcription factors within the circuit to buffer against intra- and extracellular variability. Here, we describe the design and construction of a synthetic gene oscillator in Escherichia coli that maintains a constant period over a range of temperatures. We started with a previously described synthetic dual-feedback oscillator with a temperature-dependent period. Computational modeling predicted and subsequent experiments confirmed that a single amino acid mutation to the core transcriptional repressor of the circuit results in temperature compensation. Specifically, we used a temperature-sensitive lactose repressor mutant that loses the ability to repress its target promoter at high temperatures. In the oscillator, this thermoinduction of the repressor leads to an increase in period at high temperatures that compensates for the decrease in period due to Arrhenius scaling of the reaction rates. The result is a transcriptional oscillator with a nearly constant period of 48 min for temperatures ranging from 30 °C to 41 °C. In contrast, in the absence of the mutation the period of the oscillator drops from 60 to 30 min over the same temperature range. This work demonstrates that synthetic gene circuits can be engineered to be robust to extracellular conditions through protein-level modifications.

Physical and Genetic Interactions Link Hox Function with Diverse Transcription Factors and Cell Signaling Proteins

Positional information provided by Hox homeotic transcription factors is integrated with other transcription factors and cell signaling cascades in specific combinations to dictate context- and gene-specific Hox activity. Protein-protein interactions between these groups have long been hypothesized to modulate Hox functions, yielding a context-specific function. However, difficulties in applying interaction screens to potent transcription factors have limited partner identification. A yeast two-hybrid screen using transcription activation-deficient mutants of the Drosophila melanogaster Hox protein Ultrabithorax IB identified an array of interacting proteins, consisting primarily of transcription factors and components of cell signaling pathways. Interactions were confirmed with wild-type Ultrabithorax (UBX) in phage display experiments and by immunoprecipitation for a subset of partners. In vivo assays demonstrated that two Ultrabithorax IB partners, Armadillo, regulated by Wingless/WNT signaling, and the homeodomain protein Aristaless, inhibit UBX-dependent haltere development from the default wing development pathway. Therefore, transcription factors and cell signaling proteins that subdivide Hox-specified tissues can both alter Hox function in vivo and interact with the corresponding Hox protein in vitro. UBX may also modulate partner function: the pupal death phenotype induced by ectopic expression of the UBX partner Hairy required the presence of UBX. Thus, Hox·transcription factor complexes may integrate a variety of positional cues, generating the specificity and versatility required for context-dependent Hox function.

Glycine Insertion in the Hinge Region of Lactose Repressor Protein Alters DNA Binding

Amino acid alterations were designed at the C terminus of the hinge segment (amino acids ∼51–59) that links two functional domains within lactose repressor protein (LacI). Gly was introduced between Gly58 and Lys59 to generate Gly58+1; Gln60 was changed to Gly or Pro, and up to three additional glycines were inserted following Gln60 → Gly. All mutant proteins exhibited purification behavior, CD spectra, assembly state, and inducer binding properties similar to wild-type LacI and only small differences in trypsin proteolysis patterns. In contrast, significant differences were observed in DNA binding properties. Gly58+1 exhibited a decrease of ∼100-fold in affinity for O1 operator, and sequential Gly insertion C-terminal to Gln60 → Gly resulted in progressively decreased affinity for O1operator, approaching nonspecific levels for insertion of ≥2 glycines. Where sufficient affinity for O1 operator existed, decreased binding to O1 in the presence of inducer indicated no disruption in the allosteric response for these proteins. Collectively, these results indicate that flexibility and/or spacing between the core and N-terminal domains did not significantly affect folding or assembly, but these alterations in the hinge domain profoundly altered affinity of the lactose repressor protein for its wild-type target sequence.

Tetramer opening in LacI-mediated DNA looping

Lactose repressor protein (LacI) controls transcription of the genes involved in lactose metabolism in bacteria. Essential to optimal LacI-mediated regulation is its ability to bind simultaneously to two operators, forming a loop on the intervening DNA. Recently, several lines of evidence (both theoretical and experimental) have suggested various possible loop structures associated with different DNA binding topologies and LacI tetramer structural conformations (adopted by flexing about the C-terminal tetramerization domain). We address, specifically, the role of protein opening in loop formation by employing the single-molecule tethered particle motion method on LacI protein mutants chemically cross-linked at different positions along the cleft between the two dimers. Measurements on the wild-type and uncross-linked LacI mutants led to the observation of two distinct levels of short tether length, associated with two different DNA looping structures. Restricting conformational flexibility of the protein by chemical cross-linking induces pronounced effects. Crosslinking the dimers at the level of the N-terminal DNA binding head (E36C) completely suppresses looping, whereas cross-linking near the C-terminal tetramerization domain (Q231C) results in changes of looping geometry detected by the measured tether length distributions. These observations lead to the conclusion that tetramer opening plays a definite role in at least a subset of LacI/DNA loop conformations.

Effect of lac repressor oligomerization on regulatory outcome

Regulatory outcome in a bacterial operon depends on the interactions of all the components which influence mRNA production. Levels of mRNA can be altered profoundly by both negative and positive regulatory elements which modulate initiation of transcription. The occupancy of regulatory sites on the DNA by repressors and activators is determined not only by the affinity of these proteins for their cognate site(s) but also by the oligomeric state of the regulatory protein. The lac operon in Escherichia coli provides an excellent prototypic example of the influence of protein assembly on the transcriptional status of the associated structural genes. DNA loop formation is essential for maximal repression of the lac operon and is contingent upon the presence of multiple operator sites in the DNA and the ability of the repressor to self‐associate to form a bidentate tetramer. The stability of this looped complex is enhanced significantly by DNA supercoiling. Tetramer assembly from dimers apparently occurs via interactions of a‘leucine zipper’motif in the C‐terminal domain of the protein, and the tetramer is essential to formation of looped complexes. Furthermore, analysis of the DNA‐binding characteristics of dimeric mutants has established that the monomer‐dimer association and dimer‐DNA binding (monomer does not bind to DNA) are coupled equilibria. Thus, dimer assembly is essential for generating a DNA‐binding unit, and tetramer assembly is required for formation of the stable looped DNA structure that maximally represses mRNA synthesis. Protein‐protein interactions therefore play a pivotal role in the regulatory activities of the lac repressor and must be considered when analysing the activities of any oligomeric DNA‐binding protein.

Interaction of lac repressor with inducer. Kinetic and equilibrium measurements

The rates of binding and dissociation of the inducer isopropyl-β, d -thiogalactoside to the lactose repressor protein of Escherichia coli were studied by monitoring changes in tryptophan fluorescence in a stopped-flow spectrometer. At pH 8·5 the association rate constant for inducer binding was found to be 1·7×104 m −1 s−1 and that for dissociation to be 0·53 s−1 in 1·0 m -Tris·HCl buffer. These kinetic measurements were made over a range of conditions, and a small but reproducible dependence on pH and ionic strength was observed. The presence of bound calf thymus DNA was found to affect only slightly the rats of inducer binding to the protein. The kinetically determined rate constants were used to calculate dissociation constants for the interaction between repressor and inducer. The calculated values were in agreement with those determined directly by equilibrium dialysis and ultraviolet spectral titration of repressor with inducer. It appears that repressor and inducer interact by a simple equilibrium process, the characteristics of which are similar whether repressor is free in solution or non-specifically bound to DNA.