RESUMO
Topoisomerase I (TOP1) is an essential enzyme that relaxes DNA to prevent and dissipate torsional stress during transcription. However, the mechanisms underlying the regulation of TOP1 activity remain elusive. Using enhanced cross-linking and immunoprecipitation (eCLIP) and ultraviolet-cross-linked RNA immunoprecipitation followed by total RNA sequencing (UV-RIP-seq) in human colon cancer cells along with RNA electrophoretic mobility shift assays (EMSAs), biolayer interferometry (BLI), and in vitro RNA-binding assays, we identify TOP1 as an RNA-binding protein (RBP). We show that TOP1 directly binds RNA in vitro and in cells and that most RNAs bound by TOP1 are mRNAs. Using a TOP1 RNA-binding mutant and topoisomerase cleavage complex sequencing (TOP1cc-seq) to map TOP1 catalytic activity, we reveal that RNA opposes TOP1 activity as RNA polymerase II (RNAPII) commences transcription of active genes. We further demonstrate the inhibitory role of RNA in regulating TOP1 activity by employing DNA supercoiling assays and magnetic tweezers. These findings provide insight into the coordinated actions of RNA and TOP1 in regulating DNA topological stress intrinsic to RNAPII-dependent transcription.
RESUMO
The structural organization of chromosomes is a crucial feature that defines the functional state of genes and genomes. The extent of structural changes experienced by genomes of eukaryotic cells can be dramatic and spans several orders of magnitude. At the core of these changes lies a unique group of ATPases-the SMC proteins-that act as major effectors of chromosome behavior in cells. The Smc5/6 proteins play essential roles in the maintenance of genome stability, yet their mode of action is not fully understood. Here we show that the human Smc5/6 complex recognizes unusual DNA configurations and uses the energy of ATP hydrolysis to promote their compaction. Structural analyses reveal subunit interfaces responsible for the functionality of the Smc5/6 complex and how mutations in these regions may lead to chromosome breakage syndromes in humans. Collectively, our results suggest that the Smc5/6 complex promotes genome stability as a DNA micro-compaction machine.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Instabilidade Genômica/genética , Complexos Multiproteicos/ultraestrutura , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/genética , Quebra Cromossômica , Humanos , Complexos Multiproteicos/genética , Mutação/genética , Conformação de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Structural Maintenance of Chromosomes (SMC) complexes play essential roles in genome organization across all domains of life. To determine how the activities of these large (≈50 nm) complexes are controlled by ATP binding and hydrolysis, we developed a molecular dynamics model that accounts for conformational motions of the SMC and DNA. The model combines DNA loop capture with an ATP-induced 'power stroke' to translocate the SMC complex along DNA. This process is sensitive to DNA tension: at low tension (0.1 pN), the model makes loop-capture steps of average 60 nm and up to 200 nm along DNA (larger than the complex itself), while at higher tension, a distinct inchworm-like translocation mode appears. By tethering DNA to an experimentally-observed additional binding site ('safety belt'), the model SMC complex can perform loop extrusion (LE). The dependence of LE on DNA tension is distinct for fixed DNA tension vs. fixed DNA end points: LE reversal occurs above 0.5 pN for fixed tension, while LE stalling without reversal occurs at about 2 pN for fixed end points. Our model matches recent experimental results for condensin and cohesin, and makes testable predictions for how specific structural variations affect SMC function.
Assuntos
Cromossomos , Simulação de Dinâmica Molecular , Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos/metabolismo , DNA/química , Humanos , Conformação Molecular , Translocação GenéticaRESUMO
Damaged or mismatched DNA bases result in the formation of physical defects in double-stranded DNA. In vivo, defects in DNA must be rapidly and efficiently repaired to maintain cellular function and integrity. Defects can also alter the mechanical response of DNA to bending and twisting constraints, both of which are important in defining the mechanics of DNA supercoiling. Here, we use coarse-grained molecular dynamics (MD) simulation and supporting statistical-mechanical theory to study the effect of mismatched base pairs on DNA supercoiling. Our simulations show that plectoneme pinning at the mismatch site is deterministic under conditions of relatively high force (>2 pN) and high salt concentration (>0.5 M NaCl). Under physiologically relevant conditions of lower force (0.3 pN) and lower salt concentration (0.2 M NaCl), we find that plectoneme pinning becomes probabilistic and the pinning probability increases with the mismatch size. These findings are in line with experimental observations. The simulation framework, validated with experimental results and supported by the theoretical predictions, provides a way to study the effect of defects on DNA supercoiling and the dynamics of supercoiling in molecular detail.
Assuntos
Pareamento Incorreto de Bases , DNA/química , Simulação de Dinâmica MolecularRESUMO
Eukaryote cell division features a chromosome compaction-decompaction cycle that is synchronized with their physical and topological segregation. It has been proposed that lengthwise compaction of chromatin into mitotic chromosomes via loop extrusion underlies the compaction-segregation/resolution process. We analyze this disentanglement scheme via considering the chromosome to be a succession of DNA/chromatin loops-a polymer "brush"-where active extrusion of loops controls the brush structure. Given type-II DNA topoisomerase (Topo II)-catalyzed topology fluctuations, we find that interchromosome entanglements are minimized for a certain "optimal" loop that scales with the chromosome size. The optimal loop organization is in accord with experimental data across species, suggesting an important structural role of genomic loops in maintaining a less entangled genome. Application of the model to the interphase genome indicates that active loop extrusion can maintain a level of chromosome compaction with suppressed entanglements; the transition to the metaphase state requires higher lengthwise compaction and drives complete topological segregation. Optimized genomic loops may provide a means for evolutionary propagation of gene-expression patterns while simultaneously maintaining a disentangled genome. We also find that compact metaphase chromosomes have a densely packed core along their cylindrical axes that explains their observed mechanical stiffness. Our model connects chromosome structural reorganization to topological resolution through the cell cycle and highlights a mechanism of directing Topo II-mediated strand passage via loop extrusion-driven lengthwise compaction.
Assuntos
Cromatina , Cromossomos , Animais , Cromatina/química , Cromatina/metabolismo , Cromossomos/química , Cromossomos/genética , Cromossomos/metabolismo , DNA/química , DNA/metabolismo , Genoma/genética , Humanos , Metáfase/genética , Mitose/genética , Modelos Genéticos , Schizosaccharomyces/genéticaRESUMO
Cytosine methylated at the five-carbon position is the most widely studied reversible DNA modification. Prior findings indicate that methylation can alter mechanical properties. However, those findings were qualitative and sometimes contradictory, leaving many aspects unclear. By applying single-molecule magnetic force spectroscopy techniques allowing for direct manipulation and dynamic observation of DNA mechanics and mechanically driven strand separation, we investigated how CpG and non-CpG cytosine methylation affects DNA micromechanical properties. We quantitatively characterized DNA stiffness using persistence length measurements from force-extension curves in the nanoscale length regime and demonstrated that cytosine methylation results in longer contour length and increased DNA flexibility (i.e., decreased persistence length). In addition, we observed the preferential formation of plectonemes over unwound single-stranded "bubbles" of DNA under physiologically relevant stretching forces and supercoiling densities. The flexibility and high structural stability of methylated DNA is likely to have significant consequences on the recruitment of proteins recognizing cytosine methylation and DNA packaging.
Assuntos
Citosina , DNA , DNA/metabolismo , Metilação de DNA , Micromanipulação , NanotecnologiaRESUMO
Cells possess remarkable control of the folding and entanglement topology of long and flexible chromosomal DNA molecules. It is thought that structural maintenance of chromosome (SMC) protein complexes play a crucial role in this, by organizing long DNAs into series of loops. Experimental data suggest that SMC complexes are able to translocate on DNA, as well as pull out lengths of DNA via a 'loop extrusion' process. We describe a Brownian loop-capture-ratchet model for translocation and loop extrusion based on known structural, catalytic, and DNA-binding properties of the Bacillus subtilis SMC complex. Our model provides an example of a new class of molecular motor where large conformational fluctuations of the motor 'track'-in this case DNA-are involved in the basic translocation process. Quantitative analysis of our model leads to a series of predictions for the motor properties of SMC complexes, most strikingly a strong dependence of SMC translocation velocity and step size on tension in the DNA track that it is moving along, with 'stalling' occuring at subpiconewton tensions. We discuss how the same mechanism might be used by structurally related SMC complexes (Escherichia coli MukBEF and eukaryote condensin, cohesin and SMC5/6) to organize genomic DNA.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , Modelos Químicos , Proteínas Motores Moleculares/metabolismo , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , DNA/metabolismo , Células Eucarióticas/metabolismo , Cinética , Ligação Proteica , Conformação Proteica , Estresse Mecânico , Termodinâmica , CoesinasRESUMO
The binding of transcription factors (TFs) to DNA controls most aspects of cellular function, making the understanding of their binding kinetics imperative. The standard description of bimolecular interactions posits that TF off rates are independent of TF concentration in solution. However, recent observations have revealed that proteins in solution can accelerate the dissociation of DNA-bound proteins. To study the molecular basis of facilitated dissociation (FD), we have used single-molecule imaging to measure dissociation kinetics of Fis, a key Escherichia coli TF and major bacterial nucleoid protein, from single dsDNA binding sites. We observe a strong FD effect characterized by an exchange rate [Formula: see text], establishing that FD of Fis occurs at the single-binding site level, and we find that the off rate saturates at large Fis concentrations in solution. Although spontaneous (i.e., competitor-free) dissociation shows a strong salt dependence, we find that FD depends only weakly on salt. These results are quantitatively explained by a model in which partially dissociated bound proteins are susceptible to invasion by competitor proteins in solution. We also report FD of NHP6A, a yeast TF with structure that differs significantly from Fis. We further perform molecular dynamics simulations, which indicate that FD can occur for molecules that interact far more weakly than those that we have studied. Taken together, our results indicate that FD is a general mechanism assisting in the local removal of TFs from their binding sites and does not necessarily require cooperativity, clustering, or binding site overlap.
Assuntos
DNA Fúngico/metabolismo , Proteínas HMGN/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação , DNA Fúngico/química , Proteínas HMGN/química , Cinética , Proteínas Mitocondriais/química , Ligação Proteica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/químicaRESUMO
Rebinding kinetics of molecular ligands plays a key role in the operation of biomachinery, from regulatory networks to protein transcription, and is also a key factor in design of drugs and high-precision biosensors. In this study, we investigate initial release and rebinding of ligands to their binding sites grafted on a planar surface, a situation commonly observed in single-molecule experiments and that occurs in vivo, e.g., during exocytosis. Via scaling arguments and molecular dynamic simulations, we analyze the dependence of nonequilibrium rebinding kinetics on two intrinsic length scales: the average separation distance between the binding sites and the total diffusible volume (i.e., height of the experimental reservoir in which diffusion takes place or average distance between receptor-bearing surfaces). We obtain time-dependent scaling laws for on rates and for the cumulative number of rebinding events. For diffusion-limited binding, the (rebinding) on rate decreases with time via multiple power-law regimes before the terminal steady-state (constant on-rate) regime. At intermediate times, when particle density has not yet become uniform throughout the diffusible volume, the cumulative number of rebindings exhibits a novel, to our knowledge, plateau behavior because of the three-dimensional escape process of ligands from binding sites. The duration of the plateau regime depends on the average separation distance between binding sites. After the three-dimensional diffusive escape process, a one-dimensional diffusive regime describes on rates. In the reaction-limited scenario, ligands with higher affinity to their binding sites (e.g., longer residence times) delay entry to the power-law regimes. Our results will be useful for extracting hidden timescales in experiments such as kinetic rate measurements for ligand-receptor interactions in microchannels, as well as for cell signaling via diffusing molecules.
Assuntos
Simulação de Dinâmica Molecular , Proteínas/química , Sítios de Ligação , Difusão , Cinética , Ligantes , Ligação Proteica , Conformação ProteicaRESUMO
During cell division, chromosomes must be folded into their compact mitotic form to ensure their segregation. This process is thought to be largely controlled by the action of condensin SMC protein complexes on chromatin fibers. However, how condensins organize metaphase chromosomes is not understood. We have combined micromanipulation of single human mitotic chromosomes, sub-nanonewton force measurement, siRNA interference of condensin subunit expression, and fluorescence microscopy, to analyze the role of condensin in large-scale chromosome organization. Condensin depletion leads to a dramatic (~ 10-fold) reduction in chromosome elastic stiffness relative to the native, non-depleted case. We also find that prolonged metaphase stalling of cells leads to overloading of chromosomes with condensin, with abnormally high chromosome stiffness. These results demonstrate that condensin is a main element controlling the stiffness of mitotic chromosomes. Isolated, slightly stretched chromosomes display a discontinuous condensing staining pattern, suggesting that condensins organize mitotic chromosomes by forming isolated compaction centers that do not form a continuous scaffold.
Assuntos
Adenosina Trifosfatases/fisiologia , Cromossomos Humanos/fisiologia , Proteínas de Ligação a DNA/fisiologia , Complexos Multiproteicos/fisiologia , Fenômenos Biomecânicos , Cromatina/metabolismo , Cromatina/fisiologia , Elasticidade , Humanos , Metáfase , MitoseRESUMO
Recent work indicates that twist-bend coupling plays an important role in DNA micromechanics. Here we investigate its effect on bent DNA. We provide an analytical solution of the minimum-energy shape of circular DNA, showing that twist-bend coupling induces sinusoidal twist waves. This solution is in excellent agreement with both coarse-grained simulations of minicircles and nucleosomal DNA data, which is bent and wrapped around histone proteins in a superhelical conformation. Our analysis shows that the observed twist oscillation in nucleosomal DNA, so far attributed to the interaction with the histone proteins, is an intrinsic feature of free bent DNA, and should be observable in other protein-DNA complexes.
Assuntos
DNA/química , Modelos Químicos , Nucleossomos/química , Simulação por Computador , DNA/metabolismo , DNA Circular/química , DNA Circular/metabolismo , Elasticidade , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleossomos/metabolismo , TermodinâmicaRESUMO
DNA segment exchange by site-specific serine recombinases (SRs) is thought to proceed by rigid-body rotation of the two halves of the synaptic complex, following the cleavages that create the two pairs of exchangeable ends. It remains unresolved how the amount of rotation occurring between cleavage and religation is controlled. We report single-DNA experiments for Bxb1 integrase, a model SR, where dynamics of individual synapses were observed, using relaxation of supercoiling to report on cleavage and rotation events. Relaxation events often consist of multiple rotations, with the number of rotations per relaxation event and rotation velocity sensitive to DNA sequence at the center of the recombination crossover site, torsional stress and salt concentration. Bulk and single-DNA experiments indicate that the thermodynamic stability of the annealed, but cleaved, crossover sites controls ligation efficiency of recombinant and parental synaptic complexes, regulating the number of rotations during a breakage-religation cycle. The outcome is consistent with a 'controlled rotation' model analogous to that observed for type IB topoisomerases, with religation probability varying in accord with DNA base-pairing free energies at the crossover site. Significantly, we find no evidence for a special regulatory mechanism favoring ligation and product release after a single 180° rotation.
Assuntos
DNA Nucleotidiltransferases/metabolismo , DNA/genética , DNA/metabolismo , Recombinação Genética , Proteínas Repressoras/metabolismo , Proteínas Virais/metabolismo , Sítios de Ligação Microbiológicos , Pareamento de Bases , Clivagem do DNA , Modelos Biológicos , Ligação Proteica , Especificidade por SubstratoRESUMO
We review the current understanding of the mechanics of DNA and DNA-protein complexes, from scales of base pairs up to whole chromosomes. Mechanics of the double helix as revealed by single-molecule experiments will be described, with an emphasis on the role of polymer statistical mechanics. We will then discuss how topological constraints- entanglement and supercoiling-impact physical and mechanical responses. Models for protein-DNA interactions, including effects on polymer properties of DNA of DNA-bending proteins will be described, relevant to behavior of protein-DNA complexes in vivo. We also discuss control of DNA entanglement topology by DNA-lengthwise-compaction machinery acting in concert with topoisomerases. Finally, the chapter will conclude with a discussion of relevance of several aspects of physical properties of DNA and chromatin to oncology.
Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Cromatina/química , Cromossomos , Humanos , Modelos Moleculares , Neoplasias/genética , Conformação de Ácido NucleicoRESUMO
We study a Brownian dynamics simulation model of a biopolymeric shell deformed by axial forces exerted at opposing poles. The model exhibits two distinct, linear force-extension regimes, with the response to small tensions governed by linear elasticity and the response to large tensions governed by an effective spring constant that scales with radius as R-0.25. When extended beyond the initial linear elastic regime, the shell undergoes a hysteretic, temperature-dependent buckling transition. We experimentally observe this buckling transition by stretching and imaging the lamina of isolated cell nuclei. Furthermore, the interior contents of the shell can alter mechanical response and buckling, which we show by simulating a model for the nucleus that quantitatively agrees with our micromanipulation experiments stretching individual nuclei.
Assuntos
Núcleo Celular/fisiologia , Simulação de Dinâmica Molecular , Animais , Fenômenos Biomecânicos , Cromatina/metabolismo , Elasticidade , Fibroblastos/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Modelos Lineares , Camundongos Knockout , Pirina/deficiência , Pirina/genética , TemperaturaRESUMO
We study the statistical-mechanical properties of intertwined double-helical DNAs (DNA braids). In magnetic tweezers experiments, we find that torsionally stressed stretched braids supercoil via an abrupt buckling transition, which is associated with the nucleation of a braid end loop, and that the buckled braid is characterized by a proliferation of multiple domains. Differences between the mechanics of DNA braids and supercoiled single DNAs can be understood as an effect of the increased bulkiness in the structure of the former. The experimental results are in accord with the predictions of a statistical-mechanical model.
Assuntos
DNA Super-Helicoidal/química , Conformação de Ácido Nucleico , DNA/química , MagnetismoRESUMO
We present a method of detecting sequence defects by supercoiling DNA with magnetic tweezers. The method is sensitive to a single mismatched base pair in a DNA sequence of several thousand base pairs. We systematically compare DNA molecules with 0 to 16 adjacent mismatches at 1 M monovalent salt and 3.6 pN force and show that under these conditions, a single plectoneme forms and is stably pinned at the defect. We use these measurements to estimate the energy and degree of end-loop kinking at defects. From this, we calculate the relative probability of plectoneme pinning at the mismatch under physiologically relevant conditions. Based on this estimate, we propose that DNA supercoiling could contribute to mismatch and damage sensing in vivo.
Assuntos
DNA Super-Helicoidal/química , DNA/química , Pareamento Incorreto de Bases , Pareamento de Bases , Sequência de Bases , Conformação de Ácido NucleicoRESUMO
We have found that reagents that reduce oxidized cysteines lead to destabilization of metaphase chromosome folding, suggesting that chemically linked cysteine residues may play a structural role in mitotic chromosome organization, in accord with classical studies by Dounce et al. (J Theor Biol 42:275-285, 1973) and Sumner (J Cell Sci 70:177-188, 1984a). Human chromosomes isolated into buffer unfold when exposed to dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In micromanipulation experiments which allow us to examine the mechanics of individual metaphase chromosomes, we have found that the gel-like elastic stiffness of native metaphase chromosomes is dramatically suppressed by DTT and TCEP, even before the chromosomes become appreciably unfolded. We also report protein labeling experiments on human metaphase chromosomes which allow us to tag oxidized and reduction-sensitive cysteine residues. PAGE analysis using fluorescent labels shows a small number of labeled bands. Mass spectrometry analysis of similarly labeled proteins provides a list of candidates for proteins with oxidized cysteines involved in chromosome organization, notably including components of condensin I, cohesin, the nucleosome-interacting proteins RCC1 and RCC2, as well as the RNA/DNA-binding protein NONO/p54NRB.
Assuntos
Proteínas Cromossômicas não Histona/química , Cromossomos Humanos/química , Cromossomos Humanos/ultraestrutura , Cisteína/química , Metáfase/genética , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ditiotreitol/química , Eletroforese em Gel Bidimensional , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Humanos , Cariótipo , Espectrometria de Massas , Micromanipulação , Complexos Multiproteicos/metabolismo , Notophthalmus viridescens/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição de Octâmero/metabolismo , Oxirredução , Fosfinas/química , Proteínas de Ligação a RNA/metabolismo , CoesinasRESUMO
During cell division, chromosomes are compacted in length by more than a 100-fold. A wide range of experiments demonstrated that in their compacted state, mammalian chromosomes form arrays of closely stacked consecutive â¼100 kb loops. The mechanism underlying the active process of chromosome compaction into a stack of loops is unknown. Here we test the hypothesis that chromosomes are compacted by enzymatic machines that actively extrude chromatin loops. When such loop-extruding factors (LEF) bind to chromosomes, they progressively bridge sites that are further away along the chromosome, thus extruding a loop. We demonstrate that collective action of LEFs leads to formation of a dynamic array of consecutive loops. Simulations and an analytically solved model identify two distinct steady states: a sparse state, where loops are highly dynamic but provide little compaction; and a dense state, where there are more stable loops and dramatic chromosome compaction. We find that human chromosomes operate at the border of the dense steady state. Our analysis also shows how the macroscopic characteristics of the loop array are determined by the microscopic properties of LEFs and their abundance. When the number of LEFs are used that match experimentally based estimates, the model can quantitatively reproduce the average loop length, the degree of compaction, and the general loop-array morphology of compact human chromosomes. Our study demonstrates that efficient chromosome compaction can be achieved solely by an active loop-extrusion process.
Assuntos
Cromossomos/metabolismo , Conformação de Ácido Nucleico , Algoritmos , Cromatina/metabolismo , Simulação por Computador , Humanos , Modelos Genéticos , SoftwareRESUMO
UNLABELLED: Off-rates of proteins from the DNA double helix are widely considered to be dependent only on the interactions inside the initially bound protein-DNA complex and not on the concentration of nearby molecules. However, a number of recent single-DNA experiments have shown off-rates that depend on solution protein concentration, or "facilitated dissociation." Here, we demonstrate that this effect occurs for the major Escherichia coli nucleoid protein Fis on isolated bacterial chromosomes. We isolated E. coli nucleoids and showed that dissociation of green fluorescent protein (GFP)-Fis is controlled by solution Fis concentration and exhibits an "exchange" rate constant (kexch) of ≈10(4) M(-1) s(-1), comparable to the rate observed in single-DNA experiments. We also show that this effect is strongly salt dependent. Our results establish that facilitated dissociation can be observed in vitro on chromosomes assembled in vivo IMPORTANCE: Bacteria are important model systems for the study of gene regulation and chromosome dynamics, both of which fundamentally depend on the kinetics of binding and unbinding of proteins to DNA. In experiments on isolated E. coli chromosomes, this study showed that the prolific transcription factor and chromosome packaging protein Fis displays a strong dependence of its off-rate from the bacterial chromosome on Fis concentration, similar to that observed in in vitro experiments. Therefore, the free cellular DNA-binding protein concentration can strongly affect lifetimes of proteins bound to the chromosome and must be taken into account in quantitative considerations of gene regulation. These results have particularly profound implications for transcription factors where DNA binding lifetimes can be a critical determinant of regulatory function.