ABSTRACT
Adenosine to inosine editing at the wobble position allows decoding of multiple codons by a single tRNA. This reaction is catalyzed by adenosine deaminases acting on tRNA (ADATs) and is essential for viability. In bacteria, the anticodon-specific enzyme is a homodimer that recognizes a single tRNA substrate (tRNA(Arg)(ACG)) and can efficiently deaminate short anticodon stem-loop mimics of this tRNA in vitro. The eukaryal enzyme is composed of two nonidentical subunits, ADAT2 and ADAT3, which upon heterodimerization, recognize seven to eight different tRNAs as substrates, depending on the organism, and require a full-length tRNA for activity. Although crystallographic data have provided clues to why the bacterial deaminase can utilize short substrates, residues that provide substrate binding and recognition with the eukaryotic enzymes are not currently known. In the present study, we have used a combination of mutagenesis, binding studies, and kinetic analysis to explore the contribution of individual residues in Trypanosoma brucei ADAT2 (TbADAT2) to tRNA recognition. We show that deletion of the last 10 amino acids at the C terminus of TbADAT2 abolishes tRNA binding. In addition, single alanine replacements of a string of positively charged amino acids (KRKRK) lead to binding defects that correlate with losses in enzyme activity. This region, which we have termed the KR-domain, provides a first glance at key residues involved in tRNA binding by eukaryotic tRNA editing deaminases.
Subject(s)
Adenosine Deaminase/chemistry , Adenosine Deaminase/metabolism , Protein Interaction Domains and Motifs/physiology , RNA Editing , RNA, Transfer/metabolism , Trypanosoma brucei brucei/enzymology , Trypanosoma brucei brucei/genetics , Adenosine Deaminase/genetics , Amino Acid Sequence , Enzyme Activation/genetics , Kinetics , Molecular Sequence Data , Protein Binding/genetics , Protein Binding/physiology , Protein Interaction Domains and Motifs/genetics , Protein Interaction Mapping , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , RNA Editing/physiology , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Trypanosoma brucei brucei/metabolism , Validation Studies as TopicABSTRACT
Kinetoplastids encode a single nuclear tryptophanyl tRNA that contains a CCA anticodon able to decode the UGG codons used in cytoplasmic protein synthesis but cannot decode the mitochondrial UGA codons. Following mitochondrial import, this problem is circumvented in Trypanosoma brucei by specifically editing the tRNA(Trp) anticodon to UCA, which can now decode the predominant mitochondrial UGA tryptophan codons. This tRNA also undergoes an unusual thiolation at position 33 of the anticodon loop, the only known modification at U33 in any tRNA. In other organisms, tRNA thiolation is mediated by the cysteine desulfurase, Nfs1 (IscS). However, T. brucei encodes two Nfs homologues, one cytoplasmic and the other mitochondrial. We show by a combination of RNA interference and Northern and Western analyses that the mitochondria-targeted TbNfs, and not TbNfs-like protein, is essential for thiolation of both cytosolic and mitochondrial tRNAs. Given the exclusive mitochondrial localization of TbNfs, how it mediates thiolation in the cytoplasm remains unclear. Furthermore, thiolation specifically affects thiolated tRNA stability in the cytoplasm but more surprisingly acts as a negative determinant for the essential C to U editing in T. brucei. This provides a first line of evidence for mitochondrial C to U editing regulation in this system.
