Binding of Ethidium to Yeast tRNAPhe: A New Perspective on an Old Bromide

We have reinvestigated the binding of ethidium bromide (EB) to yeast tRNAphe using frequency domain fluorometry and Global Analysis. Previous fluorescence investigations of EB — tRNA interactions, carried out for more than 30 years, have indicated a “strong” binding site with a lifetime near 26 ns and one or more “weak, non-specific” binding sites with reduced lifetimes. In our study, under specific conditions in which only one EB is bound, a fluorescence lifetime of 27 ns was obtained. However, as the EB/ tRNA ratio increased, shorter lifetime components appeared. Global Analysis of the lifetime data was consistent with a model in which the second EB molecule bound has a lifetime of only 5.4 ns. Global Analysis also indicated that this second binding event leads to a reduction in the lifetime of the first EB bound, namely from 27 ns to 17.7 ns. The lifetime decrease associated with the “strong” binding site could be due to a quenching process arising either from energy transfer between EB molecules or from alterations in the conformation of the tRNA, or both. These results are considered in light of recent NMR observations on an EB/tRNA system. We also investigated the effect of ionic strength on the lifetime and relative affinities of these two binding components and found that NaCl levels up to 900 mM did not significantly affect the results.