title: Domain movements during CCA-addition: a new function for motif C in the catalytic core of the human tRNA nucleotidyltransferases.
abstract: CCA-adding enzymes are highly specific RNA polymerases that synthesize and maintain the sequence CCA at the tRNA 3'-end. This nucleotide triplet is a prerequisite for tRNAs to be aminoacylated and to participate in protein biosynthesis. During CCA-addition, a set of highly conserved motifs in the catalytic core of these enzymes is responsible for accurate sequential nucleotide incorporation. In the nucleotide binding pocket, three amino acid residues form Watson-Crick-like base pairs to the incoming CTP and ATP. A reorientation of these templating amino acids switches the enzyme's specificity from CTP to ATP recognition. However, the mechanism underlying this essential structural rearrangement is not understood. Here, we show that motif C, whose actual function has not been identified yet, contributes to the switch in nucleotide specificity during polymerization. Biochemical characterization as well as EPR spectroscopy measurements of the human enzyme reveal that mutating the highly conserved amino acid position D139 in this motif interferes with AMP incorporation and affects interdomain movements in the enzyme. We propose a model of action, where motif C forms a flexible spring element modulating the relative orientation of the enzyme's head and body domains to accommodate the growing 3'-end of the tRNA. Furthermore, these conformational transitions initiate the rearranging of the templating amino acids to switch the specificity of the nucleotide binding pocket from CTP to ATP during CCA-synthesis.
authors: a Institute for Biochemistry; University of Leipzig ; Leipzig , Germany.
authors: Felix G M
authors: FG
authors: Ernst
authors: Christian
authors: C
authors: Rickert
authors: Alexander
authors: A
authors: Bluschke
authors: Heike
authors: H
authors: Betat
authors: Heinz-Jürgen
authors: HJ
authors: Steinhoff
authors: Mario
authors: M
authors: Mörl
mesh_terms: D020134:Catalytic Domain; D006801:Humans; D062105:Molecular Docking Simulation; D009154:Mutation; D017434:Protein Structure, Tertiary; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D012343:RNA, Transfer; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase
keywords: CCA-adding enzyme; CCA-addition; DEER; EPR spectroscopy; tRNA; tRNA maturation; tRNA nucleotidyltransferase