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 microM tRNA is used, the optimal terbium ion concentration for detecting Mg2+-induced tertiary structural changes is 50-60 microM. 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.
Hargittai
M R
MR
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis 55455, USA.
Musier-Forsyth
K
K
D001345:Autoradiography / Q000379:methods; D001483:Base Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D008274:Magnesium; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010761:Phosphorus Radioisotopes; D012343:RNA, Transfer / Q000737:chemistry*; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry*; D006163:Ribonuclease T1; D013725:Terbium
D010761:Phosphorus Radioisotopes; D012346:RNA, Transfer, Amino Acyl; D013725:Terbium; C067490:terbium chloride; D012343:RNA, Transfer; D006163:Ribonuclease T1; D008274:Magnesium