A nickel complex cleaves uridine in folded RNA structures: application to E. coli tmRNA and related engineered molecules.

Abstract : To gain more insight about Escherichia coli tmRNA structure, NiCR, a square planar macrocyclic nickel (II) complex, was used to probe guanine N7 exposure. On the basis of this additional structural information, a refined secondary structure of the molecule is proposed. In addition to its known specificity for guanine N7, we show here that the chemical probe can also cleave at specific uridine residues. In contrast to the alkaline-labile modification of guanine, the reactivity of NiCR at these uridine residues results in direct strand scission. To better characterize the uridine cleavage sites and assess the importance of the RNA structure for the reaction to occur, smaller RNA molecules derived from one pseudoknot (PK4) of E. coli tmRNA containing two uridine cleavage sites were engineered and probed. It is shown that this pseudoknot can fold by itself in solution and that the expected uridine residues are also cleaved by the nickel complex, suggesting that only a local sequence and/or structural context is required for cleavage. In E. coli tmRNA, the five uridine cleavage sites are located in double-stranded regions. These sites contain a G-U wobble base-pair and a downstream uridine which is cleaved. Using smaller RNAs derived from one stem of PK4, systematic changes in the proposed recognition motif indicate that the G-U pair is required for cleavage. Furthermore, there is no cleavage if the G-U pair is reversed. If the recognition motif is moved within the stem, the cleavage site moves accordingly. Additionally, if the recognition motif is changed such that the G-U pair is flanked by two uridine residues, the reactivity occurs only at the 3' uridine. Radical quenching studies have indicated that sulfate radical, as in the case of guanine oxidation, is involved in uridine oxidation. Although additional studies are required to better characterize the reaction, this paper reports a novel specificity for a chemical probe which may be useful for investigating structural motifs involving G-U pairs in folded RNAs.
Type de document :
Article dans une revue
Journal of Molecular Biology, Elsevier, 1998, 279 (3), pp.577-87. 〈10.1006/jmbi.1998.1813〉
Liste complète des métadonnées

Littérature citée [31 références]  Voir  Masquer  Télécharger

http://www.hal.inserm.fr/inserm-00719619
Contributeur : Annick Lefevre <>
Soumis le : vendredi 20 juillet 2012 - 12:16:35
Dernière modification le : lundi 26 mars 2018 - 14:50:54
Document(s) archivé(s) le : vendredi 16 décembre 2016 - 01:43:45

Fichier

 Accès restreint
Fichier visible le : jamais

Connectez-vous pour demander l'accès au fichier

Identifiants

Collections

Citation

Robyn Hickerson, Cristi Watkins-Sims, Cynthia Burrows, John Atkins, Raymond Gesteland, et al.. A nickel complex cleaves uridine in folded RNA structures: application to E. coli tmRNA and related engineered molecules.. Journal of Molecular Biology, Elsevier, 1998, 279 (3), pp.577-87. 〈10.1006/jmbi.1998.1813〉. 〈inserm-00719619〉

Partager

Métriques

Consultations de la notice

227