Characterization of a temperature‐sensitive mutation that impairs the function of yeast tRNA nucleotidyltransferase

Abstract

ATP(CTP) : tRNA nucleotidyltransferase catalyses the posttranscriptional addition of cytidine, cytidine and adenosine to the 3 ends of tRNAs. Previously, a temperaturesensitive phenotype in Saccharomyces cerevisiae resulting from a mutation in the CCA1 gene coding for this enzyme was identified. Here, we show that a single guanineto-adenine transition in cca1-1 generates the temperature-sensitive phenotype. Alignment of the amino acid sequence of S. cerevisiae tRNA nucleotidyltransferase with other tRNA nucleotidyltransferases for which crystal structures have been solved suggests that the resulting Glu-to-Lys substitution is in a ß-turn connecting the structurally and functionally important head and neck domains of the protein. Proteins containing Gln, His or Phe at this position were constructed to further characterize the importance of this residue in enzyme structure and function. As with the Lys variant, the Phe and His variants generate a temperature-sensitive phenotype in isogenic yeast strains, further supporting the role of this position in maintaining the structure and function of this enzyme. Comparative biophysical and biochemical characterization of both the wild-type and variant proteins indicates that amino acid substitutions at this position can result in a structural change in the protein that reduces enzyme activity (both at the permissive and non-permissive temperatures), decreases the melting temperature of the protein and alters its stability at the non-permissive temperature (37 • C).

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

“…However, the tryptophan fluorescence measurements performed according to Shan et al (see ref. 18 ) indicate a similar binding behavior for both wt and D139A mutant enzyme forms, showing that the motif C mutation has no impact on the affinity for the tRNA-CC substrate (Fig. 5).…”

“…18 Hence, an analysis based on fluorescence quenching allows at least a careful interpretation of relative tRNA binding differences between wild-type and mutant CCA-adding enzymes. 18 The human CCA-adding enzyme carries four tryptophan residues at positions 116, 211, 353, and 387. This enabled us to monitor the reduction of fluorescence upon binding of increasing amounts of tRNA-CC in the presence of 100 mM ATPa (Fig.…”

“…To compare the tRNA binding behavior between the wt HsaCCA enzyme and the D139A variant, tryptophan fluorescence spectra were measured for the unlabeled enzymes in the presence of the non-hydrolyzable NTP analogue a,b-methylene ATP (ATPa, at 100 mM; Jena Bioscience) and varying concentrations of yeast tRNA Phe -CC transcript according to Shan et al 18 using a FluoroLog (Horiba Scientific). In a total volume of 1.2 ml, the recombinant enzymes or BSA (NEB B9001S) as negative control were diluted to concentrations of 200 mM (HsaCCA wt and HsaCCA D139A) and 450 mM (BSA), respectively.…”

“…17 While the connecting motif C shows a rather low level of sequence conservation with only three predominant positions (Dx (3,4) Gx (9) R), mutations therein can cause a temperaturesensitive phenotype, indicating an important role in catalysis. 18 Yet, crystal structures show that the location of this motif in the enzyme does not support any interaction with tRNA or nucleotides in the catalytic core but helps to correctly position head (with motifs A and B) and neck domains (motif D) for proper catalysis (Fig 1B). [8][9][10]15,19 Here, we demonstrate that this motif is participating in interdomain movements required for switching the enzyme's specificity from C-to A-addition.…”

“…18,20 However, as no kinetic parameters were determined, it is impossible to directly compare these mutants with the human D139 variant. Furthermore, sequence alignments as well as structural overlays based on the crystal structures of the human and a bacterial CCA-adding enzyme from G. stearothermophilus in combination with a structural model of the yeast enzyme generated using I-TASSER software (see ref.…”