Convergent donor and acceptor substrate utilization among kinase ribozymes.
Nucleic Acids Res
; 38(19): 6785-95, 2010 Oct.
Article
em En
| MEDLINE
| ID: mdl-20511589
ABSTRACT
Accommodation of donor and acceptor substrates is critical to the catalysis of (thio)phosphoryl group transfer, but there has been no systematic study of donor nucleotide recognition by kinase ribozymes, and there is relatively little known about the structural requirements for phosphorylating internal 2'OH. To address these questions, new self-phosphorylating ribozymes were selected that utilize ATP(gammaS) or GTP(gammaS) for 2'OH (thio)phosphorylation. Eight independent sequence families were identified among 57 sequenced isolates. Kinetics, donor nucleotide recognition and secondary structures were analyzed for representatives from each family. Each ribozyme was highly specific for its cognate donor. Competition assays with nucleotide analogs showed a remarkable convergence of donor recognition requirements, with critical contributions to recognition provided by the Watson-Crick face of the nucleobase, lesser contributions from donor nucleotide ribose hydroxyls, and little or no contribution from the Hoogsteen face. Importantly, most ribozymes showed evidence of significant interaction with one or more donor phosphates, suggesting that-unlike most aptamers-these ribozymes use phosphate interactions to orient the gamma phosphate within the active site for in-line displacement. All but one of the mapped (thio)phosphorylation sites are on unpaired guanosines within internal bulges. Comparative structural analysis identified three loosely-defined consensus structural motifs for kinase ribozyme active sites.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fosfotransferases
/
RNA Catalítico
Idioma:
En
Ano de publicação:
2010
Tipo de documento:
Article