A Eubacterial Origin for the Human tRNA Nucleotidyltransferase?

Reichert, Andreas S.; Thurlow, David L.; Mörl, Mario

doi:10.1515/bc.2001.176

Cited by 41 publications

(43 citation statements)

References 28 publications

(20 reference statements)

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“…A variety of models have been proposed to explain this remarkable behavior (18 -23), but the mechanism of CCA addition remains mysterious. Moreover, although eubacterial CCA-adding enzymes and poly(A) polymerases are closely related in sequence (13,38,44), it is still difficult to predict the activity from sequence alone (38,45). We have now found that CCA-adding activity is divided between two closely related nucleotidyltransferases in Synechocystis sp.…”

Section: Cc-and A-adding Enzymes In Synechocystis and Deinococcusmentioning

confidence: 88%

Closely Related CC- and A-adding Enzymes Collaborate to Construct and Repair the 3′-Terminal CCA of tRNA in Synechocystis sp. and Deinococcus radiodurans

Tomita¹,

Weiner

2002

Journal of Biological Chemistry

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The 3-terminal CCA sequence of tRNA is faithfully constructed and repaired by the CCA-adding enzyme (ATP(CTP):tRNA nucleotidyltransferase) using CTP and ATP as substrates but no nucleic acid template. Until recently, all CCA-adding enzymes from all three kingdoms appeared to be composed of a single kind of polypeptide with dual specificity for adding both CTP and ATP; however, we recently found that in Aquifex aeolicus, which lies near the deepest root of the eubacterial 16 S rRNA-based phylogenetic tree, CCA addition represents a collaboration between closely related CCadding and A-adding enzymes (Tomita, K. and Weiner, A. M. (2001) Science 294, 1334 -1336). Here we show that in Synechocystis sp. and Deinococcus radiodurans, as in A. aeolicus, CCA is added by homologous CC-and A-adding enzymes. We also find that the eubacterial CCA-, CC-, and A-adding enzymes, as well as the related eubacterial poly(A) polymerases, each fall into phylogenetically distinct groups derived from a common ancestor. Intriguingly, the Thermatoga maritima CCA-adding enzyme groups with the A-adding enzymes, suggesting that these distinct tRNA nucleotidyltransferase activities can intraconvert over evolutionary time.The 3Ј-terminal CCA sequence (positions 74 -76 in the standard cloverleaf representation) is universally present in the tRNAs of all organisms (1) and is important for many aspects of gene expression. The CCA terminus is required for the aminoacylation of tRNA (2, 3) and for translation on the ribosome where the CCA sequences of the aminoacyl-and peptidyl-tRNA pair with the large ribosomal RNA near the peptidyltransferase center (4 -6). In eubacteria, the CCA sequence is required for the efficient maturation of the 5Ј-end of tRNA by RNase P (7,8). In eukaryotes, the CCA sequence serves as an antideterminant to block 3Ј-exonuclease activity (9), and it is essential for the export of mature tRNA from the nucleus to the cytoplasm (10, 11).The CCA-adding enzyme (ATP(CTP):tRNA nucleotidyltransferase) builds and repairs the 3Ј-terminal CCA sequence of all tRNAs (12). CCA-adding activity has been identified in all three kingdoms, suggesting conservation of function and perhaps structure throughout evolution (13). CCA-adding activity is essential in some eubacteria as well as in all archaea and eukaryotes where some or all tRNA genes do not encode CCA (14). Yet, even in organisms such as Escherichia coli where all tRNA genes do encode CCA, CCA-adding activity confers a substantial selective advantage, probably by repairing tRNAs that have been subject to errant nucleolytic attack (15).The CCA-adding enzyme belongs to the nucleotidyltransferase (NTR) 1 family, a large protein superfamily that encompasses template-dependent DNA polymerases (DNA polymerase ␤) and template-independent RNA and DNA polymerases (poly(A) polymerase, terminal deoxynucleotidyltransferase, and CCA-adding enzymes) as well as metabolic regulators (GlnB uridylyltransferase, glutamine synthase adenylyltransferase) and antibiotic resistance factors (kanamycin nucleo...

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Section: Cc-and A-adding Enzymes In Synechocystis and Deinococcusmentioning

confidence: 88%

Closely Related CC- and A-adding Enzymes Collaborate to Construct and Repair the 3′-Terminal CCA of tRNA in Synechocystis sp. and Deinococcus radiodurans

Tomita¹,

Weiner

2002

Journal of Biological Chemistry

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“…40 Point mutations were introduced into the coding sequence using the QuickChange Site-Directed Mutagenesis Kit (Stratagene).…”

Section: Construction Of Mutant Variantsmentioning

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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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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“…Accordingly, the CCA-Ntrs were suggested to be 'sorting isozymes', i.e. multiple isoforms of proteins are synthesized from a single gene and distributed to different subcellular compartments (Chen et al, 1992;Reichert et al, 2001;Keady et al, 2002;von Braun et al, 2007). It is possible that the use of multiple in-frame start codons allows for the production of variant forms of the enzyme containing different targeting information.…”

Section: The Organellar Ntr-and Cca-papsmentioning

confidence: 99%

Polyadenylation in Arabidopsis and Chlamydomonas organelles: the input of nucleotidyltransferases, poly(A) polymerases and polynucleotide phosphorylase

et al. 2009

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SUMMARYThe polyadenylation-stimulated RNA degradation pathway takes place in plant and algal organelles, yet the identities of the enzymes that catalyze the addition of the tails remain to be clarified. In a search for the enzymes responsible for adding poly(A) tails in Chlamydomonas and Arabidopsis organelles, reverse genetic and biochemical approaches were employed. The involvement of candidate enzymes including members of the nucleotidyltransferase (Ntr) family and polynucleotide phosphorylase (PNPase) was examined. For several of the analyzed nuclear-encoded proteins, mitochondrial localization was established and possible dual targeting to mitochondria and chloroplasts could be predicted. We found that certain members of the Ntr family, when expressed in bacteria, displayed poly(A) polymerase (PAP) activity and partially complemented an Escherichia coli strain lacking the endogenous PAP1 enzyme. Other Ntr proteins appeared to be specific for tRNA maturation. When the expression of PNPase was down-regulated by RNAi in Chlamydomonas, very few poly(A) tails were detected in chloroplasts for the atpB transcript, suggesting that this enzyme may be solely responsible for chloroplast polyadenylation activity in this species. Depletion of PNPase did not affect the number or sequence of mitochondrial mRNA poly(A) tails, where unexpectedly we found, in addition to polyadenylation, poly(U)-rich tails. Together, our results identify several Ntr-PAPs and PNPase in organelle polyadenylation, and reveal novel poly(U)-rich sequences in Chlamydomonas mitochondria.

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A Eubacterial Origin for the Human tRNA Nucleotidyltransferase?

Cited by 41 publications

References 28 publications

Closely Related CC- and A-adding Enzymes Collaborate to Construct and Repair the 3′-Terminal CCA of tRNA in Synechocystis sp. and Deinococcus radiodurans

Closely Related CC- and A-adding Enzymes Collaborate to Construct and Repair the 3′-Terminal CCA of tRNA in Synechocystis sp. and Deinococcus radiodurans

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

Polyadenylation in Arabidopsis and Chlamydomonas organelles: the input of nucleotidyltransferases, poly(A) polymerases and polynucleotide phosphorylase

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