Transcription factor clusters regulate genes in eukaryotic cells

Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression

Physicochemical Properties of Ion Pairs of Biological Macromolecules

Ion pairs (also known as salt bridges) of electrostatically interacting cationic and anionic moieties are important for proteins and nucleic acids to perform their function. Although numerous three-dimensional structures show ion pairs at functionally important sites of biological macromolecules and their complexes, the physicochemical properties of the ion pairs are not well understood. Crystal structures typically show a single state for each ion pair. However, recent studies have revealed the dynamic nature of the ion pairs of the biological macromolecules. Biomolecular ion pairs undergo dynamic transitions between distinct states in which the charged moieties are either in direct contact or separated by water. This dynamic behavior is reasonable in light of the fundamental concepts that were established for small ions over the last century. In this review, we introduce the physicochemical concepts relevant to the ion pairs and provide an overview of the recent advancement in biophysical research on the ion pairs of biological macromolecules

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

Deficient uracil base excision repair leads to persistent dUMP in HIV proviruses during infection of monocytes and macrophages.

Non-dividing cells of the myeloid lineage such as monocytes and macrophages are target cells of HIV that have low dNTP pool concentrations and elevated levels of dUTP, which leads to frequent incorporation of dUMP opposite to A during reverse transcription ("uracilation"). One factor determining the fate of dUMP in proviral DNA is the host cell uracil base excision repair (UBER) system. Here we explore the relative UBER capacity of monocytes (MC) and monocyte-derived macrophages (MDM) and the fate of integrated uracilated viruses in both cell types to understand the implications of viral dUMP on HIV diversification and infectivity. We find that the kinetics for MC infection is compatible with their lifetime in vivo and their near absence of hUNG2 activity is consistent with the retention of viral dUMP at high levels at least until differentiation into macrophages, where UBER becomes possible. Overexpression of human uracil DNA glycosylase in MDM prior to infection resulted in rapid removal of dUMP from HIV cDNA and near complete depletion of dUMP-containing viral copies. This finding establishes that the low hUNG2 expression level in these cells limits UBER but that hUNG2 is restrictive against uracilated viruses. In contrast, overexpression of hUNG2 after viral integration did not accelerate the excision of uracils, suggesting that they may poorly accessible in the context of chromatin. We found that viral DNA molecules with incorporated dUMP contained unique (+) strand transversion mutations that were not observed when dUMP was absent (G→T, T→A, T→G, A→C). These observations and other considerations suggest that dUMP introduces errors predominantly during (-) strand synthesis when the template is RNA. Overall, the likelihood of producing a functional virus from in vitro infection of MC is about 50-fold and 300-fold reduced as compared to MDM and activated T cells. The results implicate viral dUMP incorporation in MC and MDM as a potential viral diversification and restriction pathway during human HIV infection

AP-Endonuclease 1 Accelerates Turnover of Human 8‑Oxoguanine DNA Glycosylase by Preventing Retrograde Binding to the Abasic-Site Product

A major product of oxidative DNA damage is 8-oxoguanine. In humans, 8-oxoguanine DNA glycosylase (hOGG1) facilitates removal of these lesions, producing an abasic (AP) site in the DNA that is subsequently incised by AP-endonuclease 1 (APE1). APE1 stimulates turnover of several glycosylases by accelerating rate-limiting product release. However, there have been conflicting accounts of whether hOGG1 follows a similar mechanism. In pre-steady-state kinetic measurements, we found that addition of APE1 had no effect on the rapid burst phase of 8-oxoguanine excision by hOGG1 but accelerated steady-state turnover (<i>k</i>_cat) by ∼10-fold. The stimulation by APE1 required divalent cations, could be detected under multiple-turnover conditions using limiting concentrations of APE1, did not require flanking DNA surrounding the hOGG1 lesion site, and occurred efficiently even when the first 49 residues of APE1’s N-terminus had been deleted. Stimulation by APE1 does not involve relief from product inhibition because thymine DNA glycosylase, an enzyme that binds more tightly to AP sites than hOGG1 does, could not effectively substitute for APE1. A stimulation mechanism involving stable protein–protein interactions between free APE1 and hOGG1, or the DNA-bound forms, was excluded using protein cross-linking assays. The combined results indicate a mechanism whereby dynamic excursions of hOGG1 from the AP site allow APE1 to invade the site and rapidly incise the phosphate backbone. This mechanism, which allows APE1 to access the AP site without forming specific interactions with the glycosylase, is a simple and elegant solution to passing along unstable intermediates in base excision repair

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