Localization of the major ethidium bromide binding site on tRNA

Binding of ethidium bromide to Escherichia coli tRNA(Val) and an RNA minihelix based on the acceptor stem and T-arm of tRNA(Val) was investigated by F-19 and H-1 NMR spectroscopy of RNAs labeled with fluorine by incorporation of 5-fluorouracil. Ethidium bromide selectively intercalates into the acceptor stem of the tRNA(Val). More than one ethidium bromide binding site is found in the acceptor stem, the strongest between base pairs A6:U67 and U7:A66. F-19 and H-1 spectra of the 5-fluorouracil-substituted minihelix RNA indicate that the molecule exists in solution as a 12 base-paired stern and a single-stranded loop. Ethidium bromide no longer intercalates between base pairs corresponding to the tRNA(Val) acceptor stem in this molecule, Instead, it intercalates between base pairs at the bottom of the long stem-loop structure. These observations suggest that ethidium bromide has a preferred intercalation site close to the base of an RNA helical stem.