Mutations in Residues Involved in Zinc Binding in the Catalytic Site of Escherichia coli Threonyl-tRNA Synthetase Confer a Dominant Lethal Phenotype

Abstract

Escherichia coli threonyl-tRNA synthetase is a homodimeric protein that acts as both an enzyme and a regulator of gene expression: the protein aminoacylates tRNA Thr isoacceptors and binds to its own mRNA, inhibiting its translation. The enzyme contains a zinc atom in its active site, which is essential for the recognition of threonine. Mutations in any of the three amino acids forming the zinc-binding site inactivate the enzyme and have a dominant negative effect on growth if the corresponding genes are placed on a multicopy plasmid. We show here that this particular property is not due to the formation of inactive heterodimers, the titration of tRNA Thr by an inactive enzyme, or its misaminoacylation but is, rather, due to the regulatory function of threonyl-tRNA synthetase. Overproduction of the inactive enzyme represses the expression of the wild-type chromosomal copy of the gene to an extent incompatible with bacterial growth.

RNA-assisted catalysis in a protein enzyme: The 2′-hydroxyl of tRNA Thr A76 promotes aminoacylation by threonyl-tRNA synthetase

“…S2e). The in vivo function of the H309A mutant was tested in a complementation experiment employing a thrSnull strain of bacteria (11). Compared with the control, the H309A strain exhibited a pronounced lag phase before log phase growth and transitioned into stationary phase at significantly lower A 600 than the wild type (Fig.…”

“…Single-turnover and presteady-state kinetic assays were performed as described for HisRS (5,12). The bacterial complementation assay was performed as described (6,11). All details, including modifications and data analysis, are presented in SI Methods.…”

Threonyl‐tRNA synthetase gene, a potential target for RNAi‐based control of three rice planthoppers

“…Mutation in the active site of binding to zinc ion can hamper the growth of organisms. [26][27][28] Apart from the classic functions of protein synthesis, nonclassic functions of aaRSs also play a variety of roles in cell regulation. Mutations in aaRSs is often associated with a series of diseases, and ThrRS is no exception.…”

Roles of the Active Site Zn(II) and Residues in Substrate Discrimination by Threonyl-tRNA Synthetase: An MD and QM/MM Investigation

“…Indeed, mutation of any of the three enzyme residues (a cysteinyl and two histidyl's) ligated to the Zn(II) inactivated or inhibited the enzyme. 14,17 In general, aminoacylation as catalyzed by aaRS proceeds via a conserved substrate-assisted mechanism. 18 More specifically, a nonbridging phosphate oxygen of the aa-AMP substrate acts as the required base to abstract a proton from either the Ado76-2′-(class I) or Ado76-3′OH (class II) group of the tRNA aa .…”

“…Experimentally, several X-ray crystal structures of ThrRS with and without various ligands bound within its aminoacylation site have been obtained. ,, Notably, based in part on these structures, the aminoacylation active site was found to contain an essential Zn­(II) ion. Indeed, mutation of any of the three enzyme residues (a cysteinyl and two histidyl’s) ligated to the Zn­(II) inactivated or inhibited the enzyme. , …”