RNA structure analysis using methidiumpropyl-EDTA·Fe(II): A base-pair-specific RNA structure probe

Abstract

Methidiumpropyl-EDTA·Fe(II) [MPE·Fe(II)] in the presence of dithiothreitol, is shown to cleave phenylalanine-accepting tRNA (tRNA(Phe)) in a structure-specific fashion. Molar ratios of MPE·Fe(II) to tRNA(Phe) of <1 preferentially cleave phosphodiester bonds known to occur in double-stranded regions of the tRNA(Phe) molecule. Microdensitometric analysis of autoradiograms of MPE·Fe(II) cleavage products following gel electrophoresis reveals a correspondence between preferred sites of MPE·Fe(II) cleavage and sites in tRNA(Phe) most sensitive to cobra venom ribonuclease, a double-strand-specific endoribonuclease. Conversely, sites of cleavage by the single-strand-specific S1 nuclease correspond to those nucleotides that are least susceptible to MPE·Fe(II) hydrolysis. Sensitive helical regions in tRNA(Phe) include the dihydrouracil and the 'TΨC' stems, which cannot be detected by cobra venom ribonuclease because of steric constraints. Phosphodiester bonds within the TΨC and dihydrouracil loop regions, which are not detected by S1 nuclease under rigorously controlled digestion conditions, are revealed by inference from their relative insensitivity to MPE·Fe(II). These results demonstrate the utility of MPE·Fe(II) as a general small molecular weight probe of RNA structure, having a greater accessibility to base-paired regions than do the more bulky enzymic probes.