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

Abstract

Lanthanide metals such as terbium have previously been shown to be useful for mapping metal-binding sites in RNA. Terbium binds to the same sites on RNA as magnesium, however, with a much higher affinity. Thus, low concentrations of terbium ions can easily displace magnesium and promote phosphodiester backbone scission. At higher concentrations, terbium cleaves RNA in a sequence-independent manner, with a preference for single-stranded, non-Watson–Crick base-paired regions. Here, we show that terbium is a sensitive probe of human tRNA^Lys,3 tertiary structure and folding. When 1 μM tRNA is used, the optimal terbium ion concentration for detecting Mg²⁺-induced tertiary structural changes is 50–60 μM. 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⁺ concentrations, cleavage is restricted to nucleotides that constitute specific metal-binding pockets. This small chemical probe should also be useful for detecting protein induced structural changes in RNA.