Open AccessTertiary structure of animal tRNA Trp in solution and interaction of tRNA Trp with tryptophanyl‐tRNA synthetase
Author(s) -
GARRET Maurice,
LABOUESSE Bernard,
LITVAK Simon,
ROMBY Pascale,
EBEL Jean Pierre,
GIEGÉ Richard
Publication year - 1984
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1984.tb07882.x
Subject(s) - transfer rna , aminoacyl trna synthetase , biology , ethylnitrosourea , alkylation , biochemistry , phosphodiester bond , rna , stereochemistry , chemistry , gene , mutant , catalysis
Alkylation in beef tRNA Trp of phosphodiester bonds by ethylnitrosourea and of N‐7 in guanosines and N‐3 in cytidines by dimethyl sulfate and carbethoxylation of N‐7 in adenosines by diethyl pyrocarbonate were investigated under various conditions. This enabled us to probe the accessibility of tRNA functional groups and to investigate the structure of tRNA Trp in solution as well as its interactions with tryptophanyl‐tRNA synthetase. The phosphate reactivity towards ethylnitrosourea of unfolded tRNA was compared to that of native tRNA. The pattern of phosphate alkylation of tRNA Trp is very similar to that found with other tRNAs studied before using the same approach with protected phosphates mainly located in the D and TΨ arms. Base modification experiments showed a striking similarity in the reactivity of conserved bases known to be involved in secondary and tertiary interactions. Differences are found with yeast tRNA Phe since beef tRNA Trp showed a more stable D stem and a less stable TΨ stem. When alkylation by ethylnitrosourea was studied with the tRNA Trp · tryptophanyl‐tRNA synthetase complex we found that phosphates located at the 5′ side of the anticodon stem and in the anticodon loop were strongly protected against the reagent. The alkylation at the N‐3 position of the two cytidines in the CCA anticodon was clearly diminished in the synthetase · tRNA complex as compared with the modification in free tRNA Trp ; in contrast the two cytidines of the terminal CCA in the acceptor stem are not protected by the synthetase. The involvement of the anticodon region of tRNA Trp in the recognition process with tryptophanyl‐tRNA synthetase was confirmed in nuclease S 1 mapping experiments.