The ability of an arginine to tryptophan substitution in Saccharomyces cerevisiae tRNA nucleotidyltransferase to alleviate a temperature-sensitive phenotype suggests a role for motif C in active site organization

Goring, Mark; Leibovitch, Matthew; Gea‐Mallorquí, Ester; Karls, Shawn; Richard, Francis; Hanic-Joyce, Pamela J.; Joyce, Paul B.M.

doi:10.1016/j.bbapap.2013.07.003

Cited by 10 publications

(7 citation statements)

References 63 publications

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“…8 Whereas for most motifs a rather precise function has been identified, the more weakly conserved motif C has only been described as a connecting element for motifs B and D in the head and neck domains of the enzyme. 20 Yet, due to the presence of three highly conserved residues D, G and R, it is very likely that motif C is more than just a passive bridging element in the catalytic core. To address this question, we replaced these three individual conserved positions by alanine in the human CCA-adding enzyme.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Ernst¹,

Rickert

Bluschke³

et al. 2015

RNA Biology

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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.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Ernst¹,

Rickert

Bluschke³

et al. 2015

RNA Biology

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“…Five of the missense mutations (p.T154I, p.M158V, p.L166S, p.R190I, and p.I223T) cluster in the active site, and 2 (p.I326T and p.K416E) are in the less well-conserved C-terminal region (supplemental Figure 2). 7 Only 1 missense variant, p.I223T, occurs in the National Heart, Lung, and Blood Institute Exome Sequencing Variant Project (http://evs.gs.washington.edu/EVS/), and then only at an allele frequency of 0.0077%. We did not identify any uncommon variants in TRNT1 in 58 other unrelated probands with nonsyndromic and phenotypically distinct syndromic CSA.…”

Section: Blood 30 October 2014 X Volume 124 Number 18 Trnt1 Mutatiomentioning

confidence: 99%

“…We examined the ability of TRNT1 mutants to complement budding yeast cells harboring a temperature-sensitive mutation in CCA1 (cca1-1, originally named ts352), 7,15 and in a CCA1 deletion strain. Although expression of the wild-type human TRNT1 in the cca1-1 strain fully restored growth at a nonpermissive temperature ( Figure 1A), expression of the mutant TRNT1 alleles provided only partial rescue ( Figure 1B).…”

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confidence: 99%

Mutations in TRNT1 cause congenital sideroblastic anemia with immunodeficiency, fevers, and developmental delay (SIFD)

Chakraborty

Schmitz-Abe

Kennedy

et al. 2014

Blood

163

144

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Mutations in genes encoding proteins that are involved in mitochondrial heme synthesis, iron-sulfur cluster biogenesis, and mitochondrial protein synthesis have previously been implicated in the pathogenesis of the congenital sideroblastic anemias (CSAs). We recently described a syndromic form of CSA associated with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD). Here we demonstrate that SIFD is caused by biallelic mutations in TRNT1, the gene encoding the CCA-adding enzyme essential for maturation of both nuclear and mitochondrial transfer RNAs. Using budding yeast lacking the TRNT1 homolog, CCA1, we confirm that the patient-associated TRNT1 mutations result in partial loss of function of TRNT1 and lead to metabolic defects in both the mitochondria and cytosol, which can account for the phenotypic pleiotropy

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“…In a total volume of 20 µL, 5 pmol of 32 P-labeled tRNA Phe or tRNA Phe -CC were incubated at 20 • C in 30 mM HEPES/KOH (pH 7.6), 30 mM KCl, and 6 mM MgCl 2 for 30 to 120 min with up to 90 ng of recombinant enzyme in the presence of all four NTPs (1 mM each) or ATP (0.1 mM) and CTP (0.1 mM) separately, similar to assay conditions frequently described in the literature [22,24,25,62]. Reaction products were ethanol precipitated, separated on a 10% denaturing PAGE and visualized on a PhosphorImager (GE Healthcare).…”

Section: In Vitro Nucleotide Incorporation Assaymentioning

confidence: 99%

Divergent Evolution of Eukaryotic CC- and A-Adding Enzymes

Erber

Franz

Betat

et al. 2020

IJMS

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Synthesis of the CCA end of essential tRNAs is performed either by CCA-adding enzymes or as a collaboration between enzymes restricted to CC- and A-incorporation. While the occurrence of such tRNA nucleotidyltransferases with partial activities seemed to be restricted to Bacteria, the first example of such split CCA-adding activities was reported in Schizosaccharomyces pombe. Here, we demonstrate that the choanoflagellate Salpingoeca rosetta also carries CC- and A-adding enzymes. However, these enzymes have distinct evolutionary origins. Furthermore, the restricted activity of the eukaryotic CC-adding enzymes has evolved in a different way compared to their bacterial counterparts. Yet, the molecular basis is very similar, as highly conserved positions within a catalytically important flexible loop region are missing in the CC-adding enzymes. For both the CC-adding enzymes from S. rosetta as well as S. pombe, introduction of the loop elements from closely related enzymes with full activity was able to restore CCA-addition, corroborating the significance of this loop in the evolution of bacterial as well as eukaryotic tRNA nucleotidyltransferases. Our data demonstrate that partial CC- and A-adding activities in Bacteria and Eukaryotes are based on the same mechanistic principles but, surprisingly, originate from different evolutionary events.

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The ability of an arginine to tryptophan substitution in Saccharomyces cerevisiae tRNA nucleotidyltransferase to alleviate a temperature-sensitive phenotype suggests a role for motif C in active site organization

Cited by 10 publications

References 63 publications

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

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

Mutations in TRNT1 cause congenital sideroblastic anemia with immunodeficiency, fevers, and developmental delay (SIFD)

Divergent Evolution of Eukaryotic CC- and A-Adding Enzymes

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