Effector region of the translation elongation factor EF-Tu.GTP complex stabilizes an orthoester acid intermediate structure of aminoacyl-tRNA in a ternary complex.

Abstract

ABSTRACTtRNAvI from Escherichia coli was aminoacylated with [1-'3C]valine and its complex with Thermus thermophius elongation factor EF-Tu GTP was analyzed by 13C NMR spectroscopy. The results suggest that the aminoacyl residue of the valyl-tRNA in ternary complex with bacterial EF-Tu and GTP is not attached to tRNA by a regular ester bond to either a 2'-or 3'-hydroxyl group; instead, an intermediate orthoester acid structure with covalent linkage to both vicinal hydroxyls of the terminal adenosine-76 is formed. Mutation of arginine-59 located in the effector region of EF-Tu, a conserved residue in protein elongation factors and the a subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins), abolishes the stabilization of the orthoester acid structure of aminoacyl-tRNA.

Crystal Structure of the Ternary Complex of Phe-tRNA Phe , EF-Tu, and a GTP Analog

“…The electron density does not support a 2',3'-orthoester structure, as proposed on the basis of "C-NMR (nuclear magnetic resonance) experilnelnts on the ternary c o~n p l e~ (30). Furthermore, the ester bond is 26 A alvay from Arg5" ,which fails to support the suggestion that Arg5%ttahilizes the orthoester anion (30).…”

“…Furthermore, the ester bond is 26 A alvay from Arg5" ,which fails to support the suggestion that Arg5%ttahilizes the orthoester anion (30). Other "C-NMR experiments have not detected the existence of an orthoester (31).…”

Universal pathway for posttransfer editing reactions: Insights from the crystal structure of Tt PheRS with puromycin

“…Nevertheless, NMR studies of EF-Tu complex with tRNA reported that the protein can stabilize an orthoester intermediate between the two isomeric forms (27). QM/MM simulations of deacylation pathway for the TtPheRS complex with puromycin also testify that the labile hydroxyl will be locked into position by interactions with side chains and water molecules.…”

“…However, migration of the phenylalanine happens four orders of magnitude faster, thus suggesting that the equilibration rate depends on the chemical structure of the acyl group (31). The 2′→3′ transition proceeds via an intramolecular reaction with a formation of cyclic orthoester acid 2′,3′-intermediate of the aminoacyl residue (27). Fast transitions in 2′ ↔ 3′ isomers are associated with proton migration between hydroxyl groups accelerating in the presence of water molecules (30).…”

“…A vast amount of data implies that an aa-tRNA may exist concurrently in two 2′-O and 3′-O-aminoacylated isomers; i.e., the aminoacyl residue, upon forming an ester bond, migrates between the 2′-and 3′-hydroxyls at the 3′-terminal ribose of the tRNAs (26)(27)(28)(29). These findings trace back to results indicative of a solution equilibrium mixture of 2′ and 3′ isomers of ribose esters, including 2′(3′)-O-phenylalanyladenosine and 2′-and 3′-O-anthranoyladenosine (30,31).…”