Presence Of DNA-binding Proteins Research Articles

Current-density relation in the exclusion process with dynamic obstacles.

We investigate the totally asymmetric simple exclusion process (TASEP) in the presence of obstacles that dynamically bind and unbind from the lattice. The model is motivated by biological processes such as transcription in the presence of DNA-binding proteins. Similar models have been studied before using the mean-field approximation, but the exact relation between the particle current and density remains elusive. Here, we first show using extensive Monte Carlo simulations that the current-density relation in this model assumes a quasiparabolic form similar to that of the ordinary TASEP without obstacles. We then attempt to explain this relation using exact calculations in the limit of low and high density of particles. Our results suggest that the symmetric, quasiparabolic current-density relation arises through a nontrivial cancellation of higher-order terms, similarly as in the standard TASEP.

Phase Behavior of DNA in the Presence of DNA-Binding Proteins

To characterize the thermodynamical equilibrium of DNA chains interacting with a solution of nonspecific binding proteins, we implemented a Flory-Huggins free energy model. We explored the dependence on DNA and protein concentrations of the DNA collapse. For physiologically relevant values of the DNA-protein affinity, this collapse gives rise to a biphasic regime with a dense and a dilute phase; the corresponding phase diagram was computed. Using an approach based on Hamiltonian paths, we show that the dense phase has either a molten globule or a crystalline structure, depending on the DNA bending rigidity, which is influenced by the ionic strength. These results are valid at the thermodynamical equilibrium and therefore should be consistent with many biological processes, whose characteristic timescales range typically from 1 ms to 10 s. Our model may thus be applied to biological phenomena that involve DNA-binding proteins, such as DNA condensation with crystalline order, which occurs in some bacteria to protect their chromosome from detrimental factors; or transcription initiation, which occurs in clusters called transcription factories that are reminiscent of the dense phase characterized in this study.

Crowding on DNA as a Source of Bursts in mRNA Production

The time statistics of mRNA production is often non-Poissoinian, characterized by bursts of activity interspersed by periods of inactivity. While a multitude of models focuses on initiation dynamics as the source, burstyness is often independent of promoter sequence and its microscopic origins have remained elusive. We theoretically examine the effects of crowding on the DNA as a universal and promoter independent mechanism for generating and sustaining bursts. Using a modified version of the totally asymmetric exclusion process, we model RNAp traffic in the presence of DNA binding proteins, higher order genome structures, or other dynamic roadblocks to transcription. Through simulations, mean-field solutions, and scaling arguments we provide a state diagram showing under what conditions and to what extent crowding can be expected to induce bursts in mRNA production. We further show how roadblock dynamics introduces an effective attraction between polymerases, that counteracts diffusive spreading and sustains bursts throughout the elongation phase. Thus, by accounting for the effect of roadblocks to transcription, our model suggests that bursting is a natural side effect of crowding on the DNA.

SmAP: Manipulating DNA by Ultrasound - Single-Molecules Go Acoustic

The ability to study individual biomolecules in vitro has greatly expanded our knowledge of biological systems. Existing single-molecule techniques, such as optical and magnetic tweezers or atomic force microscopy, allow manipulation of individual biomolecules like DNA. They suffer from either being technically challenging or they offer low experimental throughput. We invented a novel lab-on-a-chip method to exert controllable forces on multiple DNA molecules simultaneously using ultrasound: single molecule Acoustical Pushing (smAP).smAP consists of a resonator integrated into a micro-fabricated fluidic chip. An acoustical pressure gradient is created homogeneously throughout the sample enabling to exert forces on DNA-tethered beads. By changing the amplitude of the driving voltage the pressure gradient can be altered, allowing sensitive control of the force applied to the DNA molecules.This approach makes it possible to apply forces up to hundreds of picoNewtons homogeneously over an area of several millimeter squared, allowing multiplexing to an unprecedented level. We validate this novel single-molecule method by recording force-distance curves of DNA molecules, both double- and single-stranded, in the presence of DNA-binding proteins. The simplicity and low cost makes smAP a widely accessible tool for biophysicists.

Complexity of cis-regulatory organization of six3aduring forebrain and eye development in zebrafish

BackgroundSix3a belongs to the SIX family of homeodomain proteins and is expressed in the most anterior neural plate at the beginning of neurogenesis in various species. Though the function of Six3a as a crucial regulator of eye and forebrain development has been thoroughly investigated, the transcriptional regulation of six3a is not well understood.ResultsTo elucidate the transcriptional regulation of six3a, we performed an in vivo reporter assay. Alignment of the 21-kb region surrounding the zebrafish six3a gene with the analogous region from different species identified several conserved non-coding modules. Transgenesis in zebrafish identified two enhancer elements and one suppressor. The D module drives the GFP reporter in the forebrain and eyes at an early stage, while the A module is responsible for the later expression. The A module also works as a repressor suppressing ectopic expression from the D module. Mutational analysis further minimized the A module to four highly conserved elements and the D module to three elements. Using electrophoresis mobility shift assays, we also provided evidence for the presence of DNA-binding proteins in embryonic nuclear extracts. The transcription factors that may occupy those highly conserved elements were also predicted.ConclusionThis study provides a comprehensive view of six3a transcription regulation during brain and eye development and offers an opportunity to establish the gene regulatory networks underlying neurogenesis in zebrafish.

Scanning Electrochemical Microscopy of DNA Monolayers Modified with Nile Blue

Scanning electrochemical microscopy (SECM) is used to probe long-range charge transport (CT) through DNA monolayers containing the redox-active Nile Blue (NB) intercalator covalently affixed at a specific location in the DNA film. At substrate potentials negative of the formal potential of covalently attached NB, the electrocatalytic reduction of Fe(CN)6(3-) generated at the SECM tip is observed only when NB is located at the DNA/solution interface; for DNA films containing NB in close proximity to the DNA/electrode interface, the electrocatalytic effect is absent. This behavior is consistent with both rapid DNA-mediated CT between the NB intercalator and the gold electrode as well as a rate-limiting electron transfer between NB and the solution phase Fe(CN)6(3-). The DNA-mediated nature of the catalytic cycle is confirmed through sequence-specific and localized detection of attomoles of TATA-binding protein, a transcription factor that severely distorts DNA upon binding. Importantly, the strategy outlined here is general and allows for the local investigation of the surface characteristics of DNA monolayers both in the absence and in the presence of DNA binding proteins. These experiments highlight the utility of DNA-modified electrodes as versatile platforms for SECM detection schemes that take advantage of CT mediated by the DNA base pair stack.

Thermodynamics and Kinetics of DNA-Protein Interactions from Single Molecule Force Spectroscopy Measurements

When single DNA molecules are stretched, the measurement of the resulting force as a function of extension has yielded new information about the physical, chemical, and biological properties of these important molecules. It has been shown that double-stranded DNA molecules undergo a force-induced melting transition at high forces. Forceextension measurements of single DNA molecules using optical tweezers allow us to measure the stability of DNA under a variety of solution conditions and in the presence of DNA binding proteins. Here we review our studies of DNA forceinduced melting in the presence of the classical single-stranded DNA binding protein, gene 32 protein. Bacteriophage T4 gene 32 protein (gp32) is a well studied representative of a large class of single-stranded DNA binding proteins, which are essential for the replication, recombination and repair of DNA. We have developed several new single molecule methods, which when applied to gp32, have led to significant new insights about this proteins structure-function relationships. We discuss a technique for measuring Kss, the association constant of these proteins to ssDNA, which we can determine over a large range of salt concentrations not available to bulk binding studies. In addition, we have measured the noncooperative association constants (Kds) of the weak but biologically-significant interaction with double-stranded DNA as a function of salt concentration for full-length protein and *I, a truncation of gp32 lacking the 48-residue C-terminal domain. Our results have led to a quantitative model for the salt dependence of protein binding, which we postulate to be regulated by a salt-dependent conformational change within the protein involving the C-terminal domain. With this new force spectroscopy technique, we have obtained binding rates and binding free energies for these interactions under a broad range of conditions. Our methodologies should have useful applications in many areas of DNA-protein interactions.

Localization of a mutant p53 response element on the Tissue Inhibitor of Metalloproteinase-3 promoter: Mutant p53 activities are distinct from wild-type

Missense mutations in the p53 gene have been observed in greater than 60% of all human tumors. Recent evidence indicates that some mutations in p53 arise as the cancer progresses from a benign tumor to a metastatic tumor and that these mutations in p53 actively contribute to the process of cancer progression. Previously, we reported that the expression of the gene encoding the tissue inhibitor of metalloproteinase-3 ( TIMP-3) is repressed in cells expressing codons 248 and 281 mutant p53 alleles. The ability of tumor-derived p53 mutants to inhibit TIMP-3 expression provides a novel mechanism for understanding how p53 mutations might contribute to tumorigenesis. Since mutant p53 is often expressed at elevated levels in a variety of cancers, the generation of cells in a tumor carrying certain mutations in p53 would cause inappropriately reduced expression of TIMP-3 and lead to elevated matrix metalloproteinase activity. We present the results of experiments that begin to determine the mechanism by which mutant p53 represses TIMP-3 gene expression. By generating deletion derivatives of the TIMP-3 promoter and testing them for expression and by performing DNA protein binding assays on the regions determined to be required for repression, we have identified elements that are essential for mutant p53-mediated transcriptional repression. These elements respond specifically to mutant but not wild type p53. While mutant p53 itself does not bind to the TIMP-3 promoter, we provide evidence for the presence of DNA binding proteins whose activity is enhanced in the presence of mutant p53.

Single molecule force spectroscopy studies of DNA denaturation by T4 gene 32 protein

Single molecule force spectroscopy is an emerging technique that can be used to measure the biophysical properties of single macromolecules such as nucleic acids and proteins. In particular, single DNA molecule stretching experiments are used to measure the elastic properties of these molecules and to induce structural transitions. We have demonstrated that double‒stranded DNA molecules undergo a force‒induced melting transition at high forces. Force–extension measurements of single DNA molecules using optical tweezers allow us to measure the stability of DNA under a variety of solution conditions and in the presence of DNA binding proteins. Here we review the evidence of DNA melting in these experiments and discuss the example of DNA force‒induced melting in the presence of the single‒stranded DNA binding protein T4 gene 32. We show that this force spectroscopy technique is a useful probe of DNA–protein interactions, which allows us to obtain binding rates and binding free energies for these interactions.

Probing Protein-DNA Interactions by Unzipping a Single DNA Double Helix

We present unzipping force analysis of protein association (UFAPA) as a novel and versatile method for detection of the position and dynamic nature of protein-DNA interactions. A single DNA double helix was unzipped in the presence of DNA-binding proteins using a feedback-enhanced optical trap. When the unzipping fork in a DNA reached a bound protein molecule we observed a dramatic increase in the tension in the DNA, followed by a sudden tension reduction. Analysis of the unzipping force throughout an unbinding “event” revealed information about the spatial location and dynamic nature of the protein-DNA complex. The capacity of UFAPA to spatially locate protein-DNA interactions is demonstrated by noncatalytic restriction mapping on a 4-kb DNA with three restriction enzymes ( BsoBI, XhoI, and EcoRI). A restriction map for a given restriction enzyme was generated with an accuracy of ∼25 bp. UFAPA also allows direct determination of the site-specific equilibrium association constant ( K A) for a DNA-binding protein. This capability is demonstrated by measuring the cation concentration dependence of K A for EcoRI binding. The measured values are in good agreement with previous measurements of K A over an intermediate range of cation concentration. These results demonstrate the potential utility of UFAPA for future studies of site-specific protein-DNA interactions.

Evidence for the presence of DNA-binding proteins involved in regulation of the gene expression of indole-3-pyruvic acid decarboxylase, a key enzyme in indole-3-acetic acid biosynthesis in Azospirillum lipoferum FS.

We isolated the ipdc gene coding for indole-3-pyruvic acid decarboxylase (IPDC), a key enzyme in the indole-3-pyruvic acid pathway for indole-3-acetic acid biosynthesis, in the plant growth-promoting rhizobacterium Azospirillum lipoferum FS. Gel mobility-shift assay showed the presence of two DNA-binding proteins that might be involved in regulation of the ipdc gene expression.

Sharp DNA bends as landmarks of protein-binding sites on straightened DNA.

We have developed a fluorescence-based method for mapping single or multiple protein-binding sites on straightened, large-size DNA molecules (> 5 kbp). In the described method, protein-DNA complexes were straightened and immobilized on a flat surface using surface tension. A fraction of the immobilized complexes displayed a sharp DNA bend with two DNA segments extending from the apex. The presence of DNA-binding proteins at the apex was verified by atomic force microscopy. The position of protein binding relative to the ends of the DNA molecule was determined by measuring the length of two DNA segments using fluorescence microscopy. We demonstrate the potential of the fluorescence-based method to localize protein-binding sites on the DNA template and to evaluate relative binding affinity. The proposed protein-binding-site mapping technique is simple and easy to perform. Practical applications include screening for DNA-binding proteins and the localization of protein-binding sites on large segments of DNA.

An evidence of presence of DNA-binding proteins in selection of dystrophic gene promoter

The expression of dystrophin gene has been found to be regulated through different promoters in different tissues. To understand the mechanism of tissue specific selection of promoter DNA-protein interaction studies were carried out using tissue specific nuclear extracts. Several tissue specific binding proteins have been detected which could have their role in selection and maintenance of tissue specificity of promoters.

Sharp boundaries demarcate the chromatin structure of a yeast heat-shock gene

In both induced and basally transcribed states, the chromatin structure of the yeast HSP82 heat-shock locus exhibits a remarkable degree of organization with respect to DNA sequence. The promoter region contains a constitutive DNase I hypersensitive site. The transcription unit is markedly sensitive to DNase I, and exhibits a sharp transition from a phased half- to a whole nucleosomal cleavage periodicity at the 3′ end. Distant upstream and downstream regions are also organized into distinct arrays of phased nucleosomes. Each array is demarcated by DNase I hypersensitive sites that display internal protected regions, suggesting the presence of DNA binding proteins. In addition, since these sites are of mononucleosomal DNA length, they may acquire a nucleosomal structure under certain environmental conditions without disrupting flanking nucleosomal phasing frames. Thus, the HSP82 locus is organized into specific, phased, chromatin structures that appear to function in transcriptional initiation, RNA polymerase passage, transcriptional termination, and the establishment of chromatin-domain microenvironments.

The effect of DNA-binding proteins on the RNA polymerase reaction

DNA-binding proteins, isolated fromBacillus subtilis grown to various stages in the process of sporulation, were incubated with a template for RNA polymerase prior to the addition of purified enzyme. In contrast to the control (no DNA-binding proteins), several of the fractions inhibited RNA polymerase activity and others had no effect; however, increased activity was observed in the presence of DNA-binding proteins from midlog or T0 cells. This stimulation of RNA polymerase activity required that the binding proteins interact with the template, since when the binding proteins and RNA polymerase were simultaneously added to the template, no stimulation of activity was observed. These results suggest that it is worth examining whether some of the DNA-binding proteins identified in this communication can function to direct RNA polymerase to specific sites on the template, and thus regulate transcription.