Single-molecule manipulation reveals supercoiling-dependent modulation of lac repressor-mediated DNA looping.
ABSTRACT: Gene expression regulation is a fundamental biological process which deploys specific sets of genomic information depending on physiological or environmental conditions. Several transcription factors (including lac repressor, LacI) are present in the cell at very low copy number and increase their local concentration by binding to multiple sites on DNA and looping the intervening sequence. In this work, we employ single-molecule manipulation to experimentally address the role of DNA supercoiling in the dynamics and stability of LacI-mediated DNA looping. We performed measurements over a range of degrees of supercoiling between -0.026 and +0.026, in the absence of axial stretching forces. A supercoiling-dependent modulation of the lifetimes of both the looped and unlooped states was observed. Our experiments also provide evidence for multiple structural conformations of the LacI-DNA complex, depending on torsional constraints. The supercoiling-dependent modulation demonstrated here adds an important element to the model of the lac operon. In fact, the complex network of proteins acting on the DNA in a living cell constantly modifies its topological and mechanical properties: our observations demonstrate the possibility of establishing a signaling pathway from factors affecting DNA supercoiling to transcription factors responsible for the regulation of specific sets of genes.
Project description:Protein-mediated DNA looping is ubiquitous in chromatin organization and gene regulation, but to what extent supercoiling or nucleoid associated proteins promote looping is poorly understood. Using the lac repressor (LacI), a paradigmatic loop-mediating protein, we measured LacI-induced looping as a function of either supercoiling or the concentration of the HU protein, an abundant nucleoid protein in Escherichia coli. Negative supercoiling to physiological levels with magnetic tweezers easily drove the looping probability from 0 to 100% in single DNA molecules under slight tension that likely exists in vivo. In contrast, even saturating (micromolar) concentrations of HU could not raise the looping probability above 30% in similarly stretched DNA or 80% in DNA without tension. Negative supercoiling is required to induce significant looping of DNA under any appreciable tension.
Project description:Escherichia coli lac repressor (LacI) is a paradigmatic transcriptional factor that controls the expression of lacZYA in the lac operon. This tetrameric protein specifically binds to the O1, O2 and O3 operators of the lac operon and forms a DNA loop to repress transcription from the adjacent lac promoter. In this article, we demonstrate that upon binding to the O1 and O2 operators at their native positions LacI constrains three (-) supercoils within the 401-bp DNA loop of the lac promoter and forms a topological barrier. The stability of LacI-mediated DNA topological barriers is directly proportional to its DNA binding affinity. However, we find that DNA supercoiling modulates the basal expression from the lac operon in E. coli. Our results are consistent with the hypothesis that LacI functions as a topological barrier to constrain free, unconstrained (-) supercoils within the 401-bp DNA loop of the lac promoter. These constrained (-) supercoils enhance LacI's DNA-binding affinity and thereby the repression of the promoter. Thus, LacI binding is superhelically modulated to control the expression of lacZYA in the lac operon under varying growth conditions.
Project description:Protein-mediated DNA looping, such as that induced by the lactose repressor (LacI) of Escherichia coli, is a well-known gene regulation mechanism. Although researchers have given considerable attention to DNA looping by LacI, many unanswered questions about this mechanism, including the role of protein flexibility, remain. Recent single-molecule observations suggest that the two DNA-binding domains of LacI are capable of splaying open about the tetramerization domain into an extended conformation. We hypothesized that if recent experiments were able to reveal the extended conformation, it is possible that such structures occurred in previous studies as well. In this study, we tested our hypothesis by reevaluating two classic in vitro binding assays using a computational rod model of DNA. The experiments and computations evaluate the looping of both linear DNA and supercoiled DNA minicircles over a broad range of DNA interoperator lengths. The computed energetic minima align well with the experimentally observed interoperator length for optimal loop stability. Of equal importance, the model reveals that the most stable loops for linear DNA occur when LacI adopts the extended conformation. In contrast, for DNA minicircles, optimal stability may arise from either the closed or the extended protein conformation depending on the degree of supercoiling and the interoperator length.
Project description:DNA looping mediated by the Lac repressor is an archetypal test case for modeling protein and DNA flexibility. Understanding looping is fundamental to quantitative descriptions of gene expression. Systematic analysis of LacI•DNA looping was carried out using a landscape of DNA constructs with lac operators bracketing an A-tract bend, produced by varying helical phasings between operators and the bend. Fluorophores positioned on either side of both operators allowed direct Förster resonance energy transfer (FRET) detection of parallel (P1) and antiparallel (A1, A2) DNA looping topologies anchored by V-shaped LacI. Combining fluorophore position variant landscapes allows calculation of the P1, A1 and A2 populations from FRET efficiencies and also reveals extended low-FRET loops proposed to form via LacI opening. The addition of isopropyl-?-D-thio-galactoside (IPTG) destabilizes but does not eliminate the loops, and IPTG does not redistribute loops among high-FRET topologies. In some cases, subsequent addition of excess LacI does not reduce FRET further, suggesting that IPTG stabilizes extended or other low-FRET loops. The data align well with rod mechanics models for the energetics of DNA looping topologies. At the peaks of the predicted energy landscape for V-shaped loops, the proposed extended loops are more stable and are observed instead, showing that future models must consider protein flexibility.
Project description:Lac repressor (LacI) binds two operator DNA sites, looping the intervening DNA. DNA molecules containing two lac operators bracketing a sequence-directed bend were previously shown to form hyperstable LacI-looped complexes. Biochemical studies suggested that orienting the operators outward relative to the bend direction (in construct 9C14) stabilizes a positively supercoiled closed form, with a V-shaped LacI, but that the most stable loop construct (11C12) is a more open form. Here, fluorescence resonance energy transfer (FRET) is measured on DNA loops, between fluorescein and TAMRA attached near the two operators, approximately 130 basepairs apart. For 9C14, efficient LacI-induced energy transfer ( approximately 74% based on donor quenching) confirms that the designed DNA shape can force the looped complex into a closed form. From enhanced acceptor emission, correcting for observed donor-dependent quenching of acceptor fluorescence, approximately 52% transfer was observed. Time-resolved FRET suggests that this complex exists in both closed- and open form populations. Less efficient transfer, approximately 10%, was detected for DNA-LacI sandwiches and 11C12-LacI, consistent with an open form loop. This demonstration of long-range FRET in large DNA loops confirms that appropriate DNA design can control loop geometry. LacI flexibility may allow it to maintain looping with other proteins bound or under different intracellular conditions.
Project description:DNA-protein loops can be essential for gene regulation. The Escherichia coli lactose (lac) operon is controlled by DNA-protein loops that have been studied for decades. Here we adapt this model to test the hypothesis that negative superhelical strain facilitates the formation of short-range (6-8 DNA turns) repression loops in E. coli. The natural negative superhelicity of E. coli DNA is regulated by the interplay of gyrase and topoisomerase enzymes, adding or removing negative supercoils, respectively. Here, we measured quantitatively DNA looping in three different E. coli strains characterized by different levels of global supercoiling: wild type, gyrase mutant (gyrB226), and topoisomerase mutant (?topA10). DNA looping in each strain was measured by assaying repression of the endogenous lac operon, and repression of ten reporter constructs with DNA loop sizes between 70-85 base pairs. Our data are most simply interpreted as supporting the hypothesis that negative supercoiling facilitates gene repression by small DNA-protein loops in living bacteria.
Project description:The Escherichia coli lactose repressor protein (LacI) provides a classic model for understanding protein-induced DNA looping. LacI has a C-terminal four-helix bundle tetramerization domain that may act as a flexible hinge. In previous work, several DNA constructs, each containing two lac operators bracketing a sequence-induced bend, were designed to stabilize different possible looping geometries. The resulting hyperstable LacI-DNA loops exist as both a compact "closed" form with a V-shaped repressor and also a more "open" form with an extended hinge. The "9C14" construct was of particular interest because footprinting, electrophoretic mobility shift, and ring closure experiments suggested that it forms both geometries. Previous fluorescence resonance energy transfer (FRET) measurements gave an efficiency of energy transfer (ET) of 70%, confirming the existence of a closed form. These measurements could not determine whether open form or intermediate geometries are populated or the timescale of interconversion. We have now applied single-molecule FRET to Cy3, Cy5 double-labeled LacI-DNA loops diffusing freely in solution. By using multiple excitation wavelengths and by carefully examining the behavior of the zero-ET peak during titration with LacI, we show that the LacI-9C14 loop exists exclusively in a single closed form exhibiting essentially 100% ET.
Project description:The lac repressor protein (LacI) efficiently represses transcription of the lac operon in Escherichia coli by binding to two distant operator sites on the bacterial DNA and causing the intervening DNA to form a loop. We employed single-molecule tethered particle motion to observe LacI-mediated loop formation and breakdown in DNA constructs that incorporate optimized operator binding sites and intrinsic curvature favorable to loop formation. Previous bulk competition assays indirectly measured the loop lifetimes in these optimized DNA constructs as being on the order of days; however, we measured these same lifetimes to be on the order of minutes for both looped and unlooped states. In a range of single-molecule DNA competition experiments, we found that the resistance of the LacI-DNA complex to competitive binding is a function of both the operator strength and the interoperator sequence. To explain these findings, we present what we believe to be a new kinetic model of loop formation and DNA competition. In this proposed new model, we hypothesize a new unlooped state in which the unbound DNA-binding domain of the LacI protein interacts nonspecifically with nonoperator DNA adjacent to the operator site at which the second LacI DNA-binding domain is bound.
Project description:Lac repressor, the first discovered transcriptional regulator, has been shown to confer multiple-modes of binding to its operator sites depending on the central spacer length. Other homolog members in the LacI/GalR family (PurR and YcjW) cannot bind their operator sites with similar structural flexibility. To decipher the underlying mechanism for this unique property, we used Spec-seq approach combined with site-directed mutagenesis to quantify the DNA binding specificity of multiple hybrids of lacI and PurR. We find that lac repressor's recognition di-residues YQ and its hinge helix loop regions are both critical for its structural flexibility. Also, specificity profiling of the whole lac operator suggests that a simple additive model from single variants suffice to predict other multivariant sites' energy reasonably well, and the genome occupancy model based on this specificity data correlates well with in vivo lac repressor binding profile.
Project description:Double-stranded DNA is a locally inflexible polymer that resists bending and twisting over hundreds of base pairs. Despite this, tight DNA bending is biologically important for DNA packaging in eukaryotic chromatin and tight DNA looping is important for gene repression in prokaryotes. We and others have previously shown that sequence nonspecific DNA kinking proteins, such as Escherichia coli heat unstable and Saccharomyces cerevisiae non-histone chromosomal protein 6A (Nhp6A), facilitate lac repressor (LacI) repression loops in E. coli. It has been unknown if this facilitation involves direct protein binding to the tightly bent DNA loop or an indirect effect promoting global negative supercoiling of DNA. Here we adapt two high-resolution in vivo protein-mapping techniques to demonstrate direct binding of the heterologous Nhp6A protein at a LacI repression loop in living E. coli cells.