ABSTRACT
Protein kinase Gcn2 attenuates protein synthesis in response to amino acid starvation while stimulating translation of a transcriptional activator of amino acid biosynthesis. Gcn2 activation requires a domain related to histidyl-tRNA synthetase (HisRS), the enzyme that aminoacylates tRNAHis. While evidence suggests that deacylated tRNA binds the HisRS domain for kinase activation, ribosomal P-stalk proteins have been implicated as alternative activating ligands on stalled ribosomes. We report crystal structures of the HisRS domain of Chaetomium thermophilum Gcn2 that reveal structural mimicry of both catalytic (CD) and anticodon-binding (ABD) domains, which in authentic HisRS bind the acceptor stem and anticodon loop of tRNAHis. Elements for forming histidyl adenylate and aminoacylation are lacking, suggesting that Gcn2HisRS was repurposed for kinase activation, consistent with mutations in the CD that dysregulate yeast Gcn2 function. Substituting conserved ABD residues well positioned to contact the anticodon loop or that form a conserved ABD-CD interface impairs Gcn2 function in starved cells. Mimicry in Gcn2HisRS of two highly conserved structural domains for binding both ends of tRNA-each crucial for Gcn2 function-supports that deacylated tRNAs activate Gcn2 and exemplifies how a metabolic enzyme is repurposed to host new local structures and sequences that confer a novel regulatory function.
Subject(s)
Chaetomium , Histidine-tRNA Ligase , Protein Serine-Threonine Kinases , Chaetomium/enzymology , Chaetomium/genetics , Chaetomium/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Histidine-tRNA Ligase/metabolism , Histidine-tRNA Ligase/chemistry , Histidine-tRNA Ligase/genetics , Stress, Physiological , Fungal Proteins/metabolism , Fungal Proteins/chemistry , Fungal Proteins/genetics , Crystallography, X-Ray , Models, Molecular , Protein Domains , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/chemistryABSTRACT
The aim of this study was to express and purify human histydyl-tRNA synthetase related gene and to prepare its polyantibody. The open reading frame was amplified by PCR, and then recombined into prokaryotic expression vector pQE30 and transformed into E. coli M15 for expression. The expressed products were induced by IPTG after the reconstructed pQE30 was transferred into M15. After purified by Ni affinity chromatography, the product was identified to be a single band by SDS-PAGE. The rabbits were inoculated with purified products. High-titer polyantibody was successfully prepared. Highly-purified expression product and prepared polyantibody may provide a good basis for further study.