Domain movements during CCA-addition: A new function for motif C in the catalytic core of the human tRNA nucleotidyltransferases

Abstract

These authors equally contributed to this work.Keywords: CCA-adding enzyme, CCA-addition, DEER, EPR spectroscopy, tRNA nucleotidyltransferase, tRNA, tRNA maturation CCA-adding enzymes are highly specific RNA polymerases that synthesize and maintain the sequence CCA at the tRNA 3 0 -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 0 -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.

Adaptation of theRomanomermis culicivoraxCCA-adding enzyme to miniaturized armless tRNA substrates

“…As controls, wt enzymes HsaCCA and RcuCCA were included. The Romanomermis enzyme showed an efficient and robust binding to mt-tRNA Ile , resulting in a K d value of 0.9 µM, while for the human enzyme, no significant binding could be detected, as previously reported for this enzyme class (Shi et al, 1998;Tretbar et al, 2011;Ernst et al, 2015). With K d values of 1.5 and 1.4 µM, both Hsa/Rcu chimeras A and B show a binding behavior similar to that of the RcuCCA wild type enzyme, demonstrating that both N-and C-termini contribute to the recognition of armless tRNAs.…”

“…As EcoCCA showed no activity on armless tRNAs, this enzyme was excluded from further analysis. To discriminate between C-and A-addition, assays were performed on tRNAs lacking the CCA-end in the presence of either α-32 P-CTP or α-32 P-ATP and unlabeled CTP (Lizano et al, 2008;Just et al, 2008;Ernst et al, 2015). As shown in table 1, k cat for CC-addition on tRNA Phe is in a similar range for both enzymes, whereas the incorporation of the terminal A residue is somewhat less efficient for RcuCCA (2-fold).…”

“…Motif A catalyzes the nucleotide transfer onto the tRNA substrate via a two metal ion mechanism (Steitz, 1998). Motif B discriminates between ribose and deoxyribose (Li et al, 2002), while motif C is a flexible element which coordinates interdomain movements, contributing to the proper orientation of the substrates within the active center (Toh et al, 2009;Ernst et al, 2015). Motif D represents an amino acid-based template, where an arginine and an aspartate residue form Watson-Crick-like hydrogen bonds with the incoming nucleotide, and their orientation in the catalytic core determines the specificity for CTP or ATP, respectively (Li et al, 2002).…”

The CCA‐adding enzyme: A central scrutinizer in tRNA quality control

“…Interestingly, this is true for both class I ( A. fulgidus ) as well as class II ( Homo sapiens ) CCA‐adding enzymes, indicating that both versions fulfill this scrutinizing function of tRNA stability. In addition, Ernst et al recently observed a similar contraction reaction in the human CCA‐adding enzyme during addition of the terminal A residue, a further indication that class II enzymes follow comparable mechanism to monitor the structural quality of the tRNA substrate . Hence, this inspection of tRNA intactness seems to represent a general quality surveillance in all kingdoms of life.…”

Divergent Evolution of Eukaryotic CC- and A-Adding Enzymes

“…Hence, it is possible that other enzyme parts important for A-addition are also affected in the CC-adding enzymes. Besides the B/A motif and the flexible loop, a springy hinge region consisting of interacting α-helical elements located in motif C helps to define the number of nucleotides to be incorporated and contributes to the structural rearrangements of the NTP binding pocket, although no direct interaction with the loop region could be demonstrated [24,33,58]. As the sequence variation of this motif between CC-and CCA-adding enzymes is identical to that found within the CCA-adding enzymes, it is highly unlikely that motif C mutations are responsible for the restricted activity of the investigated CC-adding enzymes.…”