Localization of the major ethidium bromide binding site on tRNA.

Binding of ethidium bromide to Escherichia coli tRNAVal and an RNA minihelix based on the acceptor stem and T-arm of tRNAVal was investigated by 19F and 1H NMR spectroscopy of RNAs labeled with fluorine by incorporation of 5-fluorouracil. Ethidium bromide selectively intercalates into the acceptor stem of the tRNAVal. More than one ethidium bromide binding site is found in the acceptor stem, the strongest between base pairs A6:U67 and U7:A66. 19F and 1H spectra of the 5-fluorouracil-substituted minihelix RNA indicate that the molecule exists in solution as a 12 base-paired stem and a single-stranded loop. Ethidium bromide no longer intercalates between base pairs corresponding to the tRNAVal 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.