Positive and negative impacts of nonspecific sites during target location by a sequence-specific DNA-binding protein: origin of the optimal search at physiological ionic strength

The inducible transcription factor Egr-1, which recognizes a 9-bp target DNA sequence via three zinc-finger domains, rapidly activates particular genes upon cellular stimuli such as neuronal signals and vascular stresses. Here, using the stopped-flow fluorescence method, we measured the target search kinetics of the Egr-1 zinc-finger protein at various ionic strengths between 40 and 400 mM KCl and found the most efficient search at 150 mM KCl. We further investigated the kinetics of intersegment transfer, dissociation, and sliding of this protein on DNA at distinct concentrations of KCl. Our data suggest that Egr-1's kinetic properties are well suited for efficient scanning of chromosomal DNA in vivo. Based on a newly developed theory, we analyzed the origin of the optimal search efficiency at physiological ionic strength. Target association is accelerated by nonspecific binding to nearby sites and subsequent sliding to the target as well as by intersegment transfer. Although these effects are stronger at lower ionic strengths, such conditions also favor trapping of the protein at distant nonspecific sites, decelerating the target association. Our data demonstrate that Egr-1 achieves the optimal search at physiological ionic strength through a compromise between the positive and negative impacts of nonspecific interactions with DNA

Signal Transmission in Escherichia coli Cyclic AMP Receptor Protein for Survival in Extreme Acidic Conditions

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado.During the life cycle of enteric bacterium Escherichia coli, it encounters a wide spectrum of pH changes. The asymmetric dimer of the cAMP receptor protein, CRP, plays a key role in regulating the expressions of genes and the survival of E. coli. To elucidate the pH effects on the mechanism of signal transmission, we present a combination of results derived from ITC, crystallography, and computation. CRP responds to a pH change by inducing a differential effect on the affinity for the binding events to the two cAMP molecules, ensuing in a reversible conversion between positive and negative cooperativity at high and low pH, respectively. The structures of four crystals at pH ranging from 7.8 to 6.5 show that CRP responds by inducing a differential effect on the structures of the two subunits, particularly in the DNA binding domain. Employing the COREX/BEST algorithm, computational analysis shows the change in the stability of residues at each pH. The change in residue stability alters the connectivity between residues including those in cAMP and DNA binding sites. Consequently, the differential impact on the topology of the connectivity surface among residues in adjacent subunits is the main reason for differential change in affinity; that is, the pH-induced differential change in residue stability is the biothermodynamic basis for the change in allosteric behavior. Furthermore, the structural asymmetry of this homodimer amplifies the differential impact of any perturbations. Hence, these results demonstrate that the combination of these approaches can provide insights into the underlying mechanism of an apparent complex allostery signal and transmission in CRP.National Institutes of HealthRevisión por pare

Residence Times of Molecular Complexes in Solution from NMR Data of Intermolecular Hydrogen-Bond Scalar Coupling

The residence times of molecular complexes in solution are important for understanding biomolecular functions and drug actions. We show that NMR data of intermolecular hydrogen-bond scalar couplings can yield information on the residence times of molecular complexes in solution. The molecular exchange of binding partners via the breakage and reformation of a complex causes self-decoupling of intermolecular hydrogen-bond scalar couplings, and this self-decoupling effect depends on the residence time of the complex. For protein–DNA complexes, we investigated the salt concentration dependence of intermolecular hydrogen-bond scalar couplings between the protein side-chain ¹⁵N and DNA phosphate ³¹P nuclei, from which the residence times were analyzed. The results were consistent with those obtained by ¹⁵N_<i>z</i>-exchange spectroscopy. This self-decoupling-based kinetic analysis is unique in that it does not require any different signatures for the states involved in the exchange, whereas such conditions are crucial for kinetic analyses by typical NMR and other methods

Disordered N‑Terminal Domain of Human Uracil DNA Glycosylase (hUNG2) Enhances DNA Translocation

Nuclear human uracil–DNA glycosylase (hUNG2) initiates base excision repair (BER) of genomic uracils generated through misincorporation of dUMP or through deamination of cytosines. Like many human DNA glycosylases, hUNG2 contains an unstructured N-terminal domain that encodes a nuclear localization signal, protein binding motifs, and sites for post-translational modifications. Although the N-terminal domain has minimal effects on DNA binding and uracil excision kinetics, we report that this domain enhances the ability of hUNG2 to translocate on DNA chains as compared to the catalytic domain alone. The enhancement is most pronounced when physiological ion concentrations and macromolecular crowding agents are used. These data suggest that crowded conditions in the human cell nucleus promote the interaction of the N-terminus with duplex DNA during translocation. The increased contact time with the DNA chain likely contributes to the ability of hUNG2 to locate densely spaced uracils that arise during somatic hypermutation and during fluoropyrimidine chemotherapy