David P. Mascotti

Thermodynamics of Ligand-Nucleic Acid Interactions

Ligand-and protein-DNA equilibria are extremely sensitive to solution conditions (e.g., salt, temperature, and pH), and, in general, the effects of different solution variables are interdependent (i.e., linked). As a result, an assessment of the basis for the stability and specificity of ligand-or protein-DNA interactions requires quantitative studies of these interactions as a function of a range of solution variables. Many of the most dramatic effects on the stability of these interactions result from changes in the entropy of the system, caused by the preferential interaction of small molecules, principally ions which are released into solution on complex formation. A determination of the contributions of these entropy changes to the stability and specificity of protein-and ligand-DNA interactions requires thermodynamic approaches and cannot be assessed from structural studies alone.

Nonspecific ligand-DNA equilibrium binding parameters determined by fluorescence methods

Publisher Summary This chapter focuses on the use of steady-state fluorescence techniques to monitor changes in the ligand (protein) that accompany binding, with examples drawn from studies of ligands and proteins that bind nonspecifically to nucleic acids. Spectroscopic methods—such as fluorescence, UV absorbance, and circular dichroism—offer many advantages for the study of ligand–nucleic acid as well as other macromolecular interactions. To use a change in a spectroscopic signal that is induced on formation of a ligand–nucleic acid complex to obtain a true equilibrium binding isotherm, either the relationship between the signal change and the degree of binding must be known or a method of analysis must be used that does not require knowledge of this relationship. If some relationship between the signal change and the degree of binding is assumed (e.g., linear), then the resulting binding isotherm and thermodynamic parameters determined from that binding isotherm are only as valid as the assumed relationship. The chapter also presents ligand binding density function (LBDF) analysis in which it reviews the case in which binding is accompanied by a change in the signal (fluorescence) of the ligand.

Involvement of Heme in the Degradation of Iron-regulatory Protein 2

Iron-regulatory proteins (IRPs) recognize and bind to specific RNA structures called iron-responsive elements. Mediation of these binding interactions by iron and iron-containing compounds regulates several post-transcriptional events relevant to iron metabolism. There are two known IRPs, IRP1 and IRP2, both of which can respond to iron fluxes in the cell. There is ample evidence that IRP1 is converted by iron to cytoplasmic aconitase in vivo. It has also been shown that, under certain conditions, a significant fraction of IRP1 is degraded in cells exposed to iron or heme. Studies have shown that the degradation of IRP1 that is induced by iron can be inhibited by either desferrioxamine mesylate (an iron chelator) or succinyl acetone (an inhibitor of heme synthesis), whereas the degradation induced by heme cannot. This suggests that heme rather than iron is responsible for this degradation. Several laboratories have shown that IRP2 is also degraded in cells treated with iron salts. We now show evidence suggesting that this IRP2 degradation may be mediated by heme. Thus, in experiments analogous to those used previously to study IRP1, we find that IRP2 is degraded in rabbit fibroblast cells exposed to heme or iron salts. However, as shown earlier with IRP1, both desferrioxamine mesylate and succinyl acetone will inhibit the degradation of IRP2 induced by iron but not that induced by heme.

Effects of the Ferritin Open Reading Frame on Translational Induction by Iron.

Publisher Summary Ferritin—a multimeric iron-storage protein—is evolutionarily conserved from prokaryotes to eukaryotes. It has been proposed that ferritin acts both to store iron for later use and to defend the cytosol against the generation of potentially toxic free radicals via Fenton oxygen chemistry. The regulation of ferritin synthesis is tightly coupled to changes in intracellular iron concentrations in both cultured cells and model vertebrate organisms. Cells in vertebrate organisms respond to excess iron chiefly by post-transcriptional mechanisms. Factors that modulate the effect of iron on ferritin expression are cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), nitric oxide (NO), and oxidative agents. Cytokines such as IL-1β modulate the expression of ferritin in the presence of iron. The regulation is accomplished both by transcriptional and translational mechanisms. There have been numerous reports demonstrating that the effect of ferritin iron-responsive element (IRE) on the iron inducibility of heterologous open reading frames is not as potent as on that of endogenous ferritin mRNAs. The difference in inducibility is accounted for by sequences downstream from the IRE and probably within the ferritin open reading frame (ORF). However, mRNAs that contain the ferritin ORF but lack IRE have little or no iron inducibility. This indicates a requirement for iron-responsive protein (IRP) to bind the IRE, and this interaction serves as the fundamental iron-responsive component of the translational regulatory system for ferritin.

Mechanisms for Induction and Rerepression of Ferritin Synthesis

An iron responsive element (IRE) located within the 5′ untranslated region (5′ UTR) of certain mRNAs (e.g., ferritin) serves as a binding site for a class of specific binding proteins [referred to as the iron regulatory proteins (IRPs)] which, when bound to an IRE, repress translation of those mRNAs [reviewed in Leibold and Guo (1992), Melefors and Hentze (1993), Klausner et al. (1993), Munro (1993), Theil (1993), Mascotti et al. (1995)]. Binding of an IRP to an IRE located proximally to the 5′ end of an mRNA is believed to prevent access of eIF-4F (the cap binding protein) to the 5′ cap structure, resulting in a blockage of initiation (Goossen et al. 1990; Bhasker et al. 1993; Gray and Hentze 1994). When chelatable iron levels are increased in the cell, an IRP is induced to dissociate from the IRE and allow initiation of translation of ferritin [reviewed in Leibold and Guo (1992), Melefors and Hentze (1993), Klausner et al. (1993), Munro (1993), Theil (1993)]. Messages that contain functional IREs in their 5′ UTRs code for ferritin (Leibold and Guo 1992; Melefors and Hentze 1993; Klausner et al. 1993; Munro 1993; Theil 1993), erythroid δ-aminolevulinic acid synthase (δ-ALAS) (Cox et al. 1991; Bhasker et al. 1993), mitochondrial aconitase (m-acon) (Dandekar et al. 1991), Drosophila melanogaster succinate dehydrogenase (Kohler et al. 1995; Gray et al. 1996), and possibly transferrin (Tf) (Cox and Adrian 1993; Cox et al. 1995).