Influence of tRNA tertiary structure and stability on aminoacylation by yeast aspartyl-tRNA synthetase.
AUTOR(ES)
Puglisi, J D
RESUMO
Mutations have been designed that disrupt the tertiary structure of yeast tRNA(Asp). The effects of these mutations on both tRNA structure and specific aspartylation by yeast aspartyl-tRNA synthetase were assayed. Mutations that disrupt tertiary interactions involving the D-stem or D-loop result in destabilization of the base-pairing in the D-stem, as monitored by nuclease digestion and chemical modification studies. These mutations also decrease the specificity constant (kcat/Km) for aspartylation by aspartyl-tRNA synthetase up to 10(3)-10(4) fold. The size of the T-loop also influences tRNA(Asp) structure and function; change of its T-loop to a tetraloop (-UUCG-) sequence results in a denatured D-stem and an almost 10(4) fold decrease of kcat/Km for aspartylation. The negative effects of these mutations on aspartylation activity are significantly alleviated by additional mutations that stabilize the D-stem. These results indicate that a critical role of tertiary structure in tRNA(Asp) for aspartylation is the maintenance of a base-paired D-stem.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=309063Documentos Relacionados
- Determinant nucleotides of yeast tRNA(Asp) interact directly with aspartyl-tRNA synthetase.
- The active site of yeast aspartyl-tRNA synthetase: structural and functional aspects of the aminoacylation reaction.
- Mirror image alternative interaction patterns of the same tRNA with either class I arginyl-tRNA synthetase or class II aspartyl-tRNA synthetase.
- Efficient aminoacylation of resected RNA helices by class II aspartyl-tRNA synthetase dependent on a single nucleotide.
- New photoactivatable structural and affinity probes of RNAs: specific features and applications for mapping of spermine binding sites in yeast tRNA(Asp) and interaction of this tRNA with yeast aspartyl-tRNA synthetase.
