2 Methods to Detect and Characterize Metal Ion Binding Sites in RNA

Abstract

Metal ions are inextricably associated with RNAs of any size and control their folding and activity to a large part. In order to understand RNA mechanisms, also the positioning, affinities and kinetics of metal ion binding must be known. Due to the spectroscopic silence and relatively fast exchange rates of the metal ions usually associated with RNAs, this task is extremely challenging and thus numerous methods have been developed and applied in the past. Here we provide an overview on the different metal ions and methods applied in RNA (bio)chemistry: The physical-chemical properties of important metal ions are presented and briefly discussed with respect to their application together with RNA. Each method ranging from spectroscopic over biochemical to computational approaches is briefly described also mentioning caveats that might occur during the experiment and/or interpretation of the results

NMR structure of the 5′ splice site in the group IIB intron Sc.ai5γ—conformational requirements for exon–intron recognition

“…Mg 2+ binding to RNA leads to changes in the chemical shifts (Δδ) of proton resonances near the coordinating atoms. These changes, however, can result from either metal ion coordination at the residue of the affected proton or from subtle structural changes induced by coordination of the metal (Sigel et al 2004;Erat and Sigel 2011) at an adjacent residue. Consequently, only a binding region can be defined but usually not the exact atoms involved in coordination.…”

“…The electrostatic surface potentials of d3 ′ EBS1 and d3 ′ EBS1•IBS1 were calculated with the PDB2PQR version 1.8 webserver (http://nbcr-222.ucsd.edu/pdb2pqr_1.8/) (Dolinsky et al 2004) and visualized with the APBS Tools2 plugin (Baker et al 2001) for PYMOL (http://www.pymol.org). H]-NOESY spectra recorded at 298 K as described earlier (Erat and Sigel 2011;Pechlaner and Sigel 2012). MgCl 2 was added in steps of 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 6.5, and 7 mM to d3 ′ EBS1 and in steps of 0, 0.5, 1, 2 3, 4, 5, 6, 7, 8, and 10 …”

Mg2+ions: do they bind to nucleobase nitrogens?

“…Magnesium has unique physicochemical properties ( 1 , 2 ) and is recognized as the most important divalent ion for RNA folding, structure and function ( 3 – 8 ). Next to monovalent cations and polyamines ( 9 – 11 ), the main Mg 2+ function is to counterbalance the high concentration of charged phosphate groups present in nucleic acids, but also to assist folding and function through specific binding modes.…”

Secondary structure confirmation and localization of Mg2+ ions in the mammalian CPEB3 ribozyme

“…Tb 3+ can be used to map Mg 2+ binding sites in RNA through hydrolytic cleavage experiments (Ciesiolka et al 1989;Sigel and Pyle 2003;Erat and Sigel 2011;Choudhary et al 2014). Lanthanide ions are assumed to be good mimics of Mg 2+ , since they occupy the same binding sites in RNA molecules, but bind with an at least three orders of magnitude higher affinity (Kayne and Cohn 1974;Sigel and Pyle 2003;Erat and Sigel 2011). In a Tb 3+ -induced cleavage experiment, Tb 3+ is added to a folded ribozyme, where it binds to RNA, displacing the previously bound Mg 2+ ions.…”