Mapping tRNA and 5S RNA Tertiary Structures by Charge Dependent Fe(II)-Catalyzed Cleavage

Abstract

Chemical and enzymatic footprinting experiments have made it possible to identify protein binding sites in DNA and RNA and to localize structural differences within nucleic acids to a resolution of a single base pair. We show here that by combining three reagents, Fe(II)·EDTA²·Fe(II)·EDDA and Fe²⁺, differential maps of sites in RNA that vary in their local conformation and/or charge can be constructed. Comparison of profiles with respect to controls in the absence of a counterion such as Mg²⁺ allows analysis of sites responsive to tertiary structure. A single site that is labile to metals such as Pb²⁺ exists in tRNA^Phe and a number of other tRNA's; this site is hyper-reactive to Fe(II), but not to the other probes. Scission induced by the neutral complex, Fe(II)·EDDA, offers the most general measure of surface accessibility, since its distribution about the target molecule is insensitive to charge. Enhanced cleavage by Fe(II) relative to the other agents is detected at several adjacent sites in 5S RNA consistent with conformational mobility. Protection at a series of positions in the arm formed by loops E and D with helix IV suggests further that at low temperature this arm interacts with loop A and helix I.