ABSTRACT
Leucyl-tRNA synthetase (LeuRS) is an essential RNA splicing factor for yeast mitochondrial introns. Intracellular experiments have suggested that it works in collaboration with a maturase that is encoded within the bI4 intron. RNA deletion mutants of the large bI4 intron were constructed to identify a competently folded intron for biochemical analysis. The minimized bI4 intron was active in RNA splicing and contrasts with previous proposals that the canonical core of the bI4 intron is deficient for catalysis. The activity of the minimized bI4 intron was enhanced in vitro by the presence of the bI4 maturase or LeuRS.
Subject(s)
Endoribonucleases/genetics , Introns/genetics , Leucine-tRNA Ligase/metabolism , Nucleotidyltransferases/genetics , RNA Splicing/genetics , Base Sequence , Binding Sites/genetics , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Fungal/metabolism , Electrophoresis, Polyacrylamide Gel , Endoribonucleases/metabolism , Guanosine/pharmacology , Kinetics , Leucine-tRNA Ligase/genetics , Magnesium/pharmacology , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Nucleotidyltransferases/metabolism , Protein Binding , RNA Splicing/drug effects , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Deletion , Two-Hybrid System TechniquesABSTRACT
Aminoacyl-transfer RNA (tRNA) synthetases, which catalyze the attachment of the correct amino acid to its corresponding tRNA during translation of the genetic code, are proven antimicrobial drug targets. We show that the broad-spectrum antifungal 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), in development for the treatment of onychomycosis, inhibits yeast cytoplasmic leucyl-tRNA synthetase by formation of a stable tRNA(Leu)-AN2690 adduct in the editing site of the enzyme. Adduct formation is mediated through the boron atom of AN2690 and the 2'- and 3'-oxygen atoms of tRNA's3'-terminal adenosine. The trapping of enzyme-bound tRNA(Leu) in the editing site prevents catalytic turnover, thus inhibiting synthesis of leucyl-tRNA(Leu) and consequentially blocking protein synthesis. This result establishes the editing site as a bona fide target for aminoacyl-tRNA synthetase inhibitors.
Subject(s)
Antifungal Agents/pharmacology , Boron Compounds/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Enzyme Inhibitors/pharmacology , Leucine-tRNA Ligase/antagonists & inhibitors , RNA Editing , RNA, Transfer, Leu/antagonists & inhibitors , Antifungal Agents/chemistry , Boron/chemistry , Boron Compounds/chemistry , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Drug Resistance, Fungal/genetics , Enzyme Inhibitors/chemistry , Leucine-tRNA Ligase/genetics , Leucine-tRNA Ligase/metabolism , Mutation , Protein Synthesis Inhibitors/chemistry , Protein Synthesis Inhibitors/pharmacology , RNA Editing/drug effects , RNA, Transfer, Leu/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/geneticsABSTRACT
Prolyl-tRNA synthetases (ProRSs) are unique among synthetases in that they have diverse architectures, notably the variable presence of a cis-editing domain homologous to the freestanding deacylase proteins YbaK and ProX. Here, we describe crystal structures of two bacterial ProRSs from the pathogen Enterococcus faecalis, which possesses an editing domain, and from Rhodopseudomonas palustris, which does not. We compare the overall structure and binding mode of ATP and prolyl-adenylate with those of the archael/eukaryote-type ProRS from Thermus thermophilus. Although structurally more homologous to YbaK, which preferentially hydrolyzes Cys-tRNA(Pro), the editing domain of E. faecalis ProRS possesses key elements similar to ProX, with which it shares the activity of hydrolyzing Ala-tRNA(Pro). The structures give insight into the complex evolution of ProRSs, the mechanism of editing, and structural differences between prokaryotic- and eukaryotic-type ProRSs that can be exploited for antibiotic design.
