Interstice mutations that block site-to-site translocation of a misactivated amino acid bound to a class I tRNA synthetase

Abstract

Class I aminoacyl-tRNA synthetases catalyze editing reactions that prevent ambiguity from entering the genetic code. Misactivated amino acids are translocated in cis from the active site for aminoacylation to the center for editing, located Ϸ30 Å away. Mutational analysis has functionally separated the two sites by creating mutations that disrupt the catalytic center for editing but not for aminoacylation and vice versa. What is not known is whether translocation per se can be disrupted without an effect on either catalytic center. Here we describe mutations in a presumptive ''hinge region'' of isoleucyl-tRNA synthetase that is situated between the two sites. Interstice mutations had little or no effect on either catalytic center. In contrast, the same specific mutations disrupted translocation. Thus, with these mutations all three functions, translocation, catalysis of aminoacylation, and editing, have been mutationally separated. The results are consistent with translocation involving a hinge-region conformational shift that does not perturb the two catalytic centers.C onsiderable evidence supports the concept that the universal tree of life could not be generated without the editing activities of aminoacyl-tRNA synthetases (1-3). These ancient proteins catalyze aminoacylation reactions that are the basis of the genetic code, that is, the matching of amino acids with specific nucleotide triplets imbedded in transfer RNAs (4-8). Inherent limitations to the capacity of active sites to discriminate between closely similar amino acids (9) was compensated by the introduction of a second active center where misactivated amino acids are cleared (10-15). Without this active site for editing, ambiguity is introduced into the code as demonstrated by the extensive misincorporation of amino acids into cellular proteins when editing is disrupted (1).Class I aminoacyl-tRNA synthetases such as isoleucyl-, valyl-, and leucyl-tRNA synthetase are characterized by a catalytic center based on a Rossmann nucleotide-binding fold of alternating ␤-strands and ␣-helices (6, 16-21). The fold is split by an insertion known as connective polypeptide 1 (CP1) that joins one half of the catalytic domain to the other (12,(21)(22)(23). This insertion contains the active site for editing, located Ϸ30 Å from the catalytic center for aminoacylation (11-13, 15, 21, 24). The editing reactions require specific tRNAs as cofactors (25,26). The role of the tRNA is to trigger translocation of the misactivated residue from the catalytic center to the active site for editing (27)(28)(29).Amino acids are condensed with ATP to form aminoacyl adenylates bound to their cognate tRNA synthetases in the first step of protein synthesis. Occasional errors lead to activation of the wrong amino acid such as activation of valine by isoleucyltRNA synthetase (IleRS) or activation of threonine by valyltRNA synthetase (10, 30). Editing proceeds through two distinct pathways (Fig. 1). One is designated as the pretransfer step (10, 31). Here the misactivated amino acid (i...

tRNA-dependent Aminoacyl-adenylate Hydrolysis by a Nonediting Class I Aminoacyl-tRNA Synthetase

“…The proposed model for pre-transfer editing by IleRS (and perhaps other class I tRNA synthetases) is highly complex (53,54). Pre-transfer editing is initiated with a "priming" step, whereby the misactivated amino acid is first covalently attached to tRNA and then shifted to the remote editing site.…”

Functional group recognition at the aminoacylation and editing sites of E. coli valyl-tRNA synthetase

“…Studies with the class Ia synthetases IleRS (Schmidt and Schimmel 1994;, valyl-tRNA synthetase (ValRS; , and LeuRS (Chen et al 2000;Cusack et al 2000;Mursinna et al 2001) showed that the aminoacylation and editing active sites of these enzymes are spatially separate; an ∼200-amino-acid insert in the aminoacylation site, designated CP1, is responsible for the editing activity (Schmidt and Schimmel 1995;Nureki et al 1998;Fukai et al 2000Fukai et al , 2003Lincecum et al 2003). Cognate tRNAs specifically trigger transfer of misactivated amino acids from the synthetic active site to the editing active site, accounting for the tRNA dependence of the editing reaction (Silvian et al 1999;Fukai et al 2000;Bishop et al 2002Bishop et al , 2003.…”

Leucyl-tRNA synthetase editing domain functions as a molecular rheostat to control codon ambiguity in Mycoplasma pathogens

“…In many cases, this activity seems to be enhanced in the presence of tRNA (20,21,23). Thus, in the evolution of LeuRS, IleRS, and ValRS, the original insertion of the CP1 domain likely affected fidelity via a set of mechanisms.…”

“…Both tRNA-dependent (20)(21)(22)(23) and tRNA-independent (22, 24) pretransfer editing activities have been reported. Also, clearance of the adenylate by selective ejection from the enzyme's active site into the aqueous environment has been suggested (24).…”