Cooperative Binding of Manganese (II) to Transfer RNA
TL;DR: From the bound Mn2+ electron spin resonance the authors estimate a minimal distance between the strong sites of about 1.5 nm; this shows that the binding sites are spread on the molecule and not clustered together.
Abstract: We have measured the binding of manganse ions to tRNA in the concentration range of 1 to 28 bound Mn2+ per tRNA at 23°. The free Mn2+ concentration was monitored by electron spin resonance. The Scatchard plot shows that the first four Mn2+ bind cooperatively, at a free Mn2+ concentration of 3 μM. The cooperativity presumably reflects a conformational change of tRNA. A resulting structure with 6–10 strong binding sites (ks= 1.8 × 105M−1), and 21–25 weak binding sites (kw= 5 × 103 M−1) is suggested.
From the bound Mn2+ electron spin resonance we estimate a minimal distance between the strong sites of about 1.5 nm; this shows that the binding sites are spread on the molecule and not clustered together.
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TL;DR: Results suggest certain divalent ions either participate directly in the cleavage mechanism or are specifically involved in stabilizing the tertiary structure of the hammerhead.
Abstract: A hammerhead self-cleaving domain composed of two oligoribonucleotides was used to study the role of divalent metal ions in the cleavage reaction. Cleavage rates were measured as a function of MgCl2, MnCl2, and CaCl2 concentration in the absence or presence of spermine. In the presence of spermine, the rate vs metal ion concentration curves are broader, and lower concentrations of divalent ions are necessary for catalytic activity. This suggests that spermine can promote proper folding of the hammerhead and one or more divalent ions are required for the reaction. Six additional divalent ions were tested for their ability to support hammerhead cleavage. In the absence of spermine, rapid cleavage was observed with Co2+ while very slow cleavage occurred with Sr2+ and Ba2+. No detectable specific cleavage was observed with Cd2+, Zn2+, or Pb2+. However, in the presence of 0.5 mM spermine, rapid cleavage was observed with Zn2+ and Cd2+, and the rate with Sr2+ was increased, indicating that while these three ions could not promote proper folding of the hammerhead they were able to stimulate cleavage. These results suggest certain divalent ions either participate directly in the cleavage mechanism or are specifically involved in stabilizing the tertiary structure of the hammerhead. Additionally, an altered divalent metal ion specificity was observed when a unique phosphorothioate linkage was inserted at the cleavage site. The substitution of a sulfur for a nonbridging oxygen atom substantially reduced the affinity of an important Mg2+ ion necessary for efficient cleavage. In contrast, the reaction proceeds normally with Mn2+, presumably due to its ability to coordinate with both oxygen and sulfur.(ABSTRACT TRUNCATED AT 250 WORDS)
341 citations
TL;DR: A picture of how magnesium stabilizes a particular folded form of RNA is presented and it is emphasized that diffuse binding can only be described rigorously by a model that accounts for long‐range electrostatic forces.
Abstract: Divalent cations, like magnesium, are crucial for the structural integrity and biological activity of RNA. In this article, we present a picture of how magnesium stabilizes a particular folded form of RNA. The overall stabilization of RNA by Mg2+ is given by the free energy of transferring RNA from a reference univalent salt solution to a mixed salt solution. This term has favorable energetic contributions from two distinct modes of binding: diffuse binding and site binding. In diffuse binding, fully hydrated Mg ions interact with the RNA via nonspecific long-range electrostatic interactions. In site binding, dehydrated Mg2+ interacts with anionic ligands specifically arranged by the RNA fold to act as coordinating ligands for the metal ion. Each of these modes has a strong coulombic contribution to binding; however, site binding is also characterized by substantial changes in ion solvation and other nonelectrostatic contributions. We will show how these energetic differences can be exploited to experimentally distinguish between these two classes of ions using analyses of binding polynomials. We survey a number of specific systems in which Mg2+–RNA interactions have been studied. In well-characterized systems such as certain tRNAs and some rRNA fragments these studies show that site-bound ions can play an important role in RNA stability. However, the crucial role of diffusely bound ions is also evident. We emphasize that diffuse binding can only be described rigorously by a model that accounts for long-range electrostatic forces. To fully understand the role of magnesium ions in RNA stability, theoretical models describing electrostatic forces in systems with complicated structures must be developed. © 1999 John Wiley & Sons, Inc. Biopoly 48: 113–135, 1998
322 citations
TL;DR: Different Fourier maps have revealed one major binding site for each of cobalt and managanese in the presence of magnesium, which is probably involved in the first stage of melting of the tRNA molecule, and may be critical for stabilizing the tertiary structure.
282 citations
182 citations
TL;DR: A rigorous theoretical model based on the nonlinear Poisson-Boltzmann (NLPB) equation is presented for understanding Mg(2+) binding to yeast tRNA(Phe) and it appears that the entire ensemble of electrostatically bound ions superficially mimics a few strongly coordinated ions.
140 citations
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TL;DR: In this paper, the paramagnetic resonance absorption of unstable metal complexes in aqueous solution has been explored and has been applied to the measurement of dissociation constants of such complexes.
Abstract: THE paramagnetic resonance absorption of unstable metal complexes in aqueous solution has been explored and has been applied to the measurement of dissociation constants of such complexes. The method has the obvious limitation, in common with other magnetic methods, that direct measurements are confined to those ions and molecules which are paramagnetic. On the other hand, the method, in common with other spectroscopic methods, has the advantage that it can measure directly and rapidly the concentration of the free metal ion in solution without disturbing any pre-existing equilibrium state.
218 citations
TL;DR: The primary structures of several tRNAs as well as some relationships between structure and function have been elucidated, and three-dimensional structure, specificity, and mechanism of action are the subjects of present research efforts.
Abstract: Transfer ribonucleic acids (tRNAs)1 occur in all living organisms. In biological protein synthesis they accept activated amino acids which are then transferred to growing peptide chains. With molecular weights lying between 25000 and 30000, tRNAs are easily within the reach of today's physical, chemical, and biochemical methods. The primary structures of several tRNAs as well as some relationships between structure and function have been elucidated. Three-dimensional structure, specificity, and mechanism of action are the subjects of present research efforts.
192 citations
01 Jan 1949
185 citations
159 citations
TL;DR: It has been found that for unfractionated tRNA of Escherichia coli and purified tRNA Phe of E. coli there is one class of interacting strong binding sites representing 0.08 site per phosphate, n 1, characterized by an enhancement factor, ϵ b 1, of 19.2 ± 1.0.
100 citations