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Titolo:
Thiophilic metal ion rescue of phosphorothioate interference within the Tetrahymena ribozyme P4-P6 domain
Autore:
Basu, S; Strobel, SA;
Indirizzi:
Yale Univ, Dept Chem, Dept Mol Biophys & Biochem, New Haven, CT 06520 USA Yale Univ New Haven CT USA 06520 ophys & Biochem, New Haven, CT 06520 USA
Titolo Testata:
RNA-A PUBLICATION OF THE RNA SOCIETY
fascicolo: 11, volume: 5, anno: 1999,
pagine: 1399 - 1407
SICI:
1355-8382(199911)5:11<1399:TMIROP>2.0.ZU;2-J
Fonte:
ISI
Lingua:
ENG
Soggetto:
PHENYLALANINE TRANSFER-RNA; GROUP-I INTRON; TERTIARY STRUCTURE; HAMMERHEAD RIBOZYME; PHOSPHATE OXYGENS; CLEAVAGE REACTION; DNA-POLYMERASE; BINDING-SITES; P RNA; CATALYSIS;
Keywords:
group I intron; metal binding; RNA folding;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
43
Recensione:
Indirizzi per estratti:
Indirizzo: Strobel, SA Yale Univ, Dept Chem, Dept Mol Biophys & Biochem, POB 208114,260 Whitney Ave, New Haven, CT 06520 USA Yale Univ POB 208114,260 Whitney Ave New Haven CT USA 06520 SA
Citazione:
S. Basu e S.A. Strobel, "Thiophilic metal ion rescue of phosphorothioate interference within the Tetrahymena ribozyme P4-P6 domain", RNA, 5(11), 1999, pp. 1399-1407

Abstract

Bivalent metal ions are essential for the folding and catalytic activitiesof many RNAs, A commonly employed biochemical technique to identify metal-binding sites in RNA is the rescue of R-P alpha-phosphorothioate (PS) interference by the addition of soft divalent metal ions, To access the ability of such experiments to accurately identify metal-ion coordinations within acomplex RNA fold, we report metal-rescue results from the Tetrahymena group I intron P4-P6 domain, where the location and coordination of five divalent metal ions have been determined by X-ray crystallography [J.H. Gate et al,, Nat Struct Biol, 1997, 4:553], We used a native gel mobility-shift to assay for P4-P6 folding in the presence of various divalent metal ions, and found that even moderate concentrations of Mn2+ (greater than or equal to 0.5 mM) can rescue PS interference at sites that do not coordinate metal ions within the P4-P6 crystal structure, To control for such effects, 2'-deoxynucleotide interference was used to titrate the Mn2+ concentration to a level that produces metal-ion-specific rescue (0.3 mM), This concentration of Mn2+ specifically rescued four of the six metal-dependent phosphorothioate effects within the RNA domain, including PS interference resulting from outer-sphere coordination to the metals, Both sites that were not specificallyrescued make inner-sphere metal-ion coordinations. Cd2+ and Zn2+ afforded rescue at a smaller subset of the six metal-specific PS sites, though againphosphates making outer-sphere coordinations to metal ions were rescued preferentially. These data on P4-P6 domain folding reinforce the need for caution when interpreting metal-rescue experiments.

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Documento generato il 03/04/20 alle ore 12:47:33