Use of terbium as a probe of tRNA tertiary structure and folding
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 tRNALys,3 tertiary structure and folding. When 1 [mu]M tRNA is used, the optimal terbium ion concentration for detecting Mg2+-induced tertiary structural changes is 50–60 [mu]M. Using these concentrations of RNA and terbium, a magnesium-dependent folding transition with a midpoint (KMg) of 2.6 mM is observed for unmodified human tRNALys,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.(Received July 11 2000)
(Revised August 16 2000)
(Accepted August 29 2000)
Key Words: lanthanides; magnesium; metal binding; RNA cleavage; tRNALys,3.
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