RNA



METHOD

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


MICHELE R. SEFFERNICK  HARGITTAI a1 and KARIN  MUSIER-FORSYTH a1a2c1
a1 Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA
a2 Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA

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 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.

Correspondence:
c1 Reprint requests to: Karin Musier-Forsyth, University of Minnesota, Department of Chemistry, 207 Pleasant Street S.E., Minneapolis, Minnesota 55455, USA; e-mail: musier@chem.umn.edu.