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Divergent Evolution of Eukaryotic CC- and A-Adding EnzymesErber, Lieselotte, Franz, Paul, Betat, Heike, Prohaska, Sonja, Mörl, Mario 26 January 2024 (has links)
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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Adaptation of the Romanomermis culicivorax CCA-Adding Enzyme to Miniaturized Armless tRNA SubstratesHennig, Oliver, Philipp, Susanne, Bonin, Sonja, Rollet, Kévin, Kolberg, Tim, Jühling, Tina, Betat, Heike, Sauter, Claude, Mörl, Mario 10 January 2024 (has links)
The mitochondrial genome of the nematode Romanomermis culicivorax encodes for
miniaturized hairpin-like tRNA molecules that lack D- as well as T-arms, strongly deviating from
the consensus cloverleaf. The single tRNA nucleotidyltransferase of this organism is fully active on
armless tRNAs, while the human counterpart is not able to add a complete CCA-end. Transplanting
single regions of the Romanomermis enzyme into the human counterpart, we identified a beta-turn
element of the catalytic core that—when inserted into the human enzyme—confers full CCA-adding
activity on armless tRNAs. This region, originally identified to position the 30
-end of the tRNA
primer in the catalytic core, dramatically increases the enzyme’s substrate affinity. While conventional
tRNA substrates bind to the enzyme by interactions with the T-arm, this is not possible in the case of
armless tRNAs, and the strong contribution of the beta-turn compensates for an otherwise too weak
interaction required for the addition of a complete CCA-terminus. This compensation demonstrates
the remarkable evolutionary plasticity of the catalytic core elements of this enzyme to adapt to
unconventional tRNA substrates.
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A Temporal Order in 5'- and 3'- Processing of Eukaryotic tRNAHis:Pöhler, Marie-Theres, Roach, Tracy M., Betat, Heike, Jackman, Jane E., Mörl, Mario 11 January 2024 (has links)
For flawless translation of mRNA sequence into protein, tRNAs must undergo a series
of essential maturation steps to be properly recognized and aminoacylated by aminoacyl-tRNA
synthetase, and subsequently utilized by the ribosome. While all tRNAs carry a 30
-terminal CCA
sequence that includes the site of aminoacylation, the additional 50
-G-1 position is a unique feature
of most histidine tRNA species, serving as an identity element for the corresponding synthetase.
In eukaryotes including yeast, both 30
-CCA and 50
-G-1 are added post-transcriptionally by tRNA
nucleotidyltransferase and tRNAHis guanylyltransferase, respectively. Hence, it is possible that
these two cytosolic enzymes compete for the same tRNA. Here, we investigate substrate preferences
associated with CCA and G-1-addition to yeast cytosolic tRNAHis, which might result in a temporal
order to these important processing events. We show that tRNA nucleotidyltransferase accepts
tRNAHis transcripts independent of the presence of G-1; however, tRNAHis guanylyltransferase
clearly prefers a substrate carrying a CCA terminus. Although many tRNA maturation steps can
occur in a rather random order, our data demonstrate a likely pathway where CCA-addition precedes
G-1 incorporation in S. cerevisiae. Evidently, the 30
-CCA triplet and a discriminator position A73 act
as positive elements for G-1 incorporation, ensuring the fidelity of G-1 addition.
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Genotyping bacterial and fungal pathogens using sequence variation in the gene for the CCA-adding enzymeFranz, Paul, Betat, Heike, Mörl, Mario 15 June 2016 (has links) (PDF)
Background: To allow an immediate treatment of an infection with suitable antibiotics and bactericides or fungicides, there is an urgent need for fast and precise identification of the causative human pathogens. Methods based on DNA sequence comparison like 16S rRNA analysis have become standard tools for pathogen verification. However, the distinction of closely related organisms remains a challenging task. To overcome such limitations, we identified a new genomic target sequence located in the single copy gene for tRNA nucleotidyltransferase fulfilling the requirements for a ubiquitous, yet highly specific DNA marker. In the present study, we demonstrate that this sequence marker has a higher discriminating potential than commonly used genotyping markers in pro- as well as eukaryotes, underscoring its applicability as an excellent diagnostic tool in infectology. Results: Based on phylogenetic analyses, a region within the gene for tRNA nucleotidyltransferase (CCA-adding enzyme) was identified as highly heterogeneous. As prominent examples for pro- and eukaryotic pathogens, several Vibrio and Aspergillus species were used for genotyping and identification in a multiplex PCR approach followed by gel electrophoresis and fluorescence-based product detection. Compared to rRNA analysis, the selected gene region of the tRNA nucleotidyltransferase revealed a seven to 30-fold higher distinction potential between closely related Vibrio or Aspergillus species, respectively. The obtained data exhibit a superb genome specificity in the diagnostic analysis. Even in the presence of a 1,000-fold excess of human genomic DNA, no unspecific amplicons were produced. Conclusions: These results indicate that a relatively short segment of the coding region for tRNA nucleotidyltransferase has a higher discriminatory potential than most established diagnostic DNA markers. Besides identifying microbial pathogens in infections, further possible applications of this new marker are food hygiene controls or metagenome analyses.
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Genotyping bacterial and fungal pathogens using sequence variation in the gene for the CCA-adding enzymeFranz, Paul, Betat, Heike, Mörl, Mario January 2016 (has links)
Background: To allow an immediate treatment of an infection with suitable antibiotics and bactericides or fungicides, there is an urgent need for fast and precise identification of the causative human pathogens. Methods based on DNA sequence comparison like 16S rRNA analysis have become standard tools for pathogen verification. However, the distinction of closely related organisms remains a challenging task. To overcome such limitations, we identified a new genomic target sequence located in the single copy gene for tRNA nucleotidyltransferase fulfilling the requirements for a ubiquitous, yet highly specific DNA marker. In the present study, we demonstrate that this sequence marker has a higher discriminating potential than commonly used genotyping markers in pro- as well as eukaryotes, underscoring its applicability as an excellent diagnostic tool in infectology. Results: Based on phylogenetic analyses, a region within the gene for tRNA nucleotidyltransferase (CCA-adding enzyme) was identified as highly heterogeneous. As prominent examples for pro- and eukaryotic pathogens, several Vibrio and Aspergillus species were used for genotyping and identification in a multiplex PCR approach followed by gel electrophoresis and fluorescence-based product detection. Compared to rRNA analysis, the selected gene region of the tRNA nucleotidyltransferase revealed a seven to 30-fold higher distinction potential between closely related Vibrio or Aspergillus species, respectively. The obtained data exhibit a superb genome specificity in the diagnostic analysis. Even in the presence of a 1,000-fold excess of human genomic DNA, no unspecific amplicons were produced. Conclusions: These results indicate that a relatively short segment of the coding region for tRNA nucleotidyltransferase has a higher discriminatory potential than most established diagnostic DNA markers. Besides identifying microbial pathogens in infections, further possible applications of this new marker are food hygiene controls or metagenome analyses.
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