Use of a novel Förster resonance energy transfer method to identify locations of site-bound metal ions in the U2-U6 snRNA complex.
U2 and U6 snRNAs pair to form a phylogenetically conserved complex at the catalytic core of the spliceosome. Interactions with divalent metal ions, particularly Mg(II), at specific sites are essential for its folding and catalytic activity. We used a novel Förster resonance energy transfer (FRET) method between site-bound luminescent lanthanide ions and a covalently attached fluorescent dye, combined with supporting stoichiometric and mutational studies, to determine locations of site-bound Tb(III) within the human U2-U6 complex. At pH 7.2, we detected three metal-ion-binding sites in: (1) the consensus ACACAGA sequence, which forms the internal loop between helices I and III; (2) the four-way junction, which contains the conserved AGC triad; and (3) the internal loop of the U6 intra-molecular stem loop (ISL). Binding at each of these sites is supported by previous phosphorothioate substitution studies and, in the case of the ISL site, by NMR. Binding of Tb(III) at the four-way junction and the ISL sites was found to be pH-dependent, with no ion binding observed below pH 6 and 7, respectively. This pH dependence of metal ion binding suggests that the local environment may play a role in the binding of metal ions, which may impact on splicing activity.
Yuan
Faqing
F
Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA.
Griffin
Laura
L
Phelps
LauraJane
L
Buschmann
Volker
V
Weston
Kenneth
K
Greenbaum
Nancy L
NL
D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002232:Carbocyanines / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000378:metabolism; D013725:Terbium / Q000737:chemistry*
D002232:Carbocyanines; D005456:Fluorescent Dyes; D007477:Ions; D012342:RNA, Small Nuclear; C405484:U2 small nuclear RNA; C405486:U6 small nuclear RNA; C089187:cyanine dye 3; D013725:Terbium