Abstract
Ferritin messenger RNA has been shown to be translationally inactivated by the binding of a cytosolic protein to a 28-nucleotide iron-responsive element (IRE) located in the 5'-untranslated region of the mRNA. This interaction has been studied using quantitative receptor-ligand binding methods with gel retardation and nitrocellulose filter binding assays for the separation of bound complex from free RNA. In competition assays the entire 5'-untranslated region and the isolated IRE bound identically. The specificity of the RNA binding was studied using IRE variants. Two IREs from transferrin receptor mRNA and several variants with single base substitutions in the stem or loop had similar affinities. RNAs which could not form a stem-loop structure bound 1000-fold less well. These studies demonstrate the importance of the RNA conformation and the relative insensitivity of binding to much of the primary sequence. Saturation assays with increasing concentrations of 32P-IRE resulted in a binding hyperbola characteristic of mass action binding to a single class of sites with a KD = 0.09 nM. At 37 degrees C the dissociation rate is 0.04 min-1 (t 1/2 = 17 min). This rate is fast enough to account for the shift of ferritin RNA from the ribonucleoprotein pool to polysomes after rats are injected with iron. Determination of the concentration of the repressor requires accounting for three interconverting pools: free active repressor, mRNA-bound protein, and inactive (low affinity) repressor. Rat liver cytosol has a concentration of free active repressor of about 1 pmol/mg protein. Protein bound to endogenous mRNA can be measured by pretreatment with micrococcal nuclease or by separation with DEAE-Sepharose chromatography; it is present at a level similar to that of the free active protein. Inclusion of high levels of thiol reductants in the binding incubations reduces the inactive or low affinity repressor, forming unstably activated protein which has the same KD as the endogenous active protein; this inactive or low affinity protein is 2-4 times more abundant. A mechanism for iron regulation is proposed which accounts for the kinetics, the multiple protein pools, and the characteristics of the protein in these pools.
Highlights
021 I I, the $Toxicology Program, Massachusetts and the )/Liver Unit, King’s College School of Medicine and Dentistry, Research Institute Denmark
Dissociation Rate of the RNA-Protein Complex-Dissociation of Rl bound to iron-responsive element (IRE)-BP in DEAE flow-through after the addition of excess cold ligand occurred with a km1 = 0.041 + 0.003 min-’ at 37 “C (n = 2) or a tlh = 17 min (Fig. 4) as determined by nitrocellulose filter binding assay
To determine the mechanism by which iron modulates the translation of ferritin mRNA and possibly other proteins of iron metabolism, it is necessary to characterize the interaction of the IRE-BP with its RNA binding site and determine the changes that occur in cellular iron levels
Summary
Vol 265, No 12, Issue of April 25, pp. 7000-7008, 1990 Printed in U.S.A. Determinants of the Interaction between the Iron-responsive Elementbinding Protein and Its Binding Site in Rat L-Ferritin mRNA*. 02139, Ferritin messenger RNA has been shown to be translationally inactivated by the binding of a cytosolic protein to a 2S-nucleotide iron-responsive element (IRE) located in the 5’-untranslated region of the mRNA. This interaction has been studied using quantitative receptor-ligand binding methods with gel retardation and nitrocellulose filter binding assays for the separation of bound complex from free RNA. RNAs which could not form a stem-loop structure bound lOOO-fold less well These studies demonstrate the importance of the RNA conformation and the relative insensitivity of binding to much of the primary sequence
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