Use of terbium as a probe of tRNA tertiary structure and folding

Lanthanide metals such as terbium have previously been shown to be useful f or mapping metal-binding sites in RNA, Terbium binds to the same sites on R NA as magnesium, however, with a much higher affinity. Thus, low concentrat ions of terbium ions can easily displace magnesium and promote phosphodiest er backbone scission. At higher concentrations, terbium cleaves RNA in a se quence-independent manner, with a preference for single-stranded, non-Watso n-Crick base-paired regions. Here, we show that terbium is a sensitive prob e of human tRNA(Lys,3) tertiary structure and folding, When 1 muM tRNA is u sed, the optimal terbium ion concentration for detecting Mg2+-induced terti ary structural changes is 50-60 muM. Using these concentrations of RNA and terbium, a magnesium-dependent folding transition with a midpoint (K-Mg) of 2.6 mM is observed for unmodified human tRNA(Lys,3), At lower Tb3+ concent rations, cleavage is restricted to nucleotides that constitute specific met al-binding pockets. This small chemical probe should also be useful for det ecting protein induced structural changes in RNA.