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. 1993 Jan 11;21(1):41-9.
doi: 10.1093/nar/21.1.41.

Influence of tRNA tertiary structure and stability on aminoacylation by yeast aspartyl-tRNA synthetase

J D Puglisi  1 , J Pütz, C Florentz, R Giegé
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Free PMC article

Influence of tRNA tertiary structure and stability on aminoacylation by yeast aspartyl-tRNA synthetase

J D Puglisi et al. Nucleic Acids Res. .
Free PMC article

Abstract

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.

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