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19 pages, 2168 KB

Open AccessArticle

Adaptation of the Romanomermis culicivorax CCA-Adding Enzyme to Miniaturized Armless tRNA Substrates

by Oliver Hennig, Susanne Philipp, Sonja Bonin, Kévin Rollet, Tim Kolberg, Tina Jühling, Heike Betat, Claude Sauter and Mario Mörl

Int. J. Mol. Sci. 2020, 21(23), 9047; https://doi.org/10.3390/ijms21239047 - 28 Nov 2020

Cited by 13 | Viewed by 3445

Abstract

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 3′-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. Full article

(This article belongs to the Section Molecular Biology)

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16 pages, 2648 KB

Open AccessArticle

Unusual Occurrence of Two Bona-Fide CCA-Adding Enzymes in Dictyostelium discoideum

by Lieselotte Erber, Anne Hoffmann, Jörg Fallmann, Monica Hagedorn, Christian Hammann, Peter F. Stadler, Heike Betat, Sonja Prohaska and Mario Mörl

Int. J. Mol. Sci. 2020, 21(15), 5210; https://doi.org/10.3390/ijms21155210 - 23 Jul 2020

Cited by 5 | Viewed by 4139

Abstract

Dictyostelium discoideum, the model organism for the evolutionary supergroup of Amoebozoa, is a social amoeba that, upon starvation, undergoes transition from a unicellular to a multicellular organism. In its genome, we identified two genes encoding for tRNA nucleotidyltransferases. Such pairs of tRNA nucleotidyltransferases usually represent collaborating partial activities catalyzing CC- and A-addition to the tRNA 3′-end, respectively. In D. discoideum, however, both enzymes exhibit identical activities, representing bona-fide CCA-adding enzymes. Detailed characterization of the corresponding activities revealed that both enzymes seem to be essential and are regulated inversely during different developmental stages of D. discoideum. Intriguingly, this is the first description of two functionally equivalent CCA-adding enzymes using the same set of tRNAs and showing a similar distribution within the cell. This situation seems to be a common feature in Dictyostelia, as other members of this phylum carry similar pairs of tRNA nucleotidyltransferase genes in their genome. Full article

(This article belongs to the Section Molecular Biology)

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18 pages, 3279 KB

Open AccessArticle

Divergent Evolution of Eukaryotic CC- and A-Adding Enzymes

by Lieselotte Erber, Paul Franz, Heike Betat, Sonja Prohaska and Mario Mörl

Int. J. Mol. Sci. 2020, 21(2), 462; https://doi.org/10.3390/ijms21020462 - 10 Jan 2020

Cited by 4 | Viewed by 4642

Abstract

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. Full article

(This article belongs to the Section Molecular Biology)

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17 pages, 3056 KB

Open AccessArticle

Origin of the Genetic Code Is Found at the Transition between a Thioester World of Peptides and the Phosphoester World of Polynucleotides

by Hyman Hartman and Temple F. Smith

Life 2019, 9(3), 69; https://doi.org/10.3390/life9030069 - 22 Aug 2019

Cited by 30 | Viewed by 7788

Abstract

The early metabolism arising in a Thioester world gave rise to amino acids and their simple peptides. The catalytic activity of these early simple peptides became instrumental in the transition from Thioester World to a Phosphate World. This transition involved the appearances of sugar phosphates, nucleotides, and polynucleotides. The coupling of the amino acids and peptides to nucleotides and polynucleotides is the origin for the genetic code. Many of the key steps in this transition are seen in the catalytic cores of the nucleotidyltransferases, the class II tRNA synthetases (aaRSs) and the CCA adding enzyme. These catalytic cores are dominated by simple beta hairpin structures formed in the Thioester World. The code evolved from a proto-tRNA, a tetramer XCCA interacting with a proto-aminoacyl-tRNA synthetase (aaRS) activating Glycine and Proline. The initial expanded code is found in the acceptor arm of the tRNA, the operational code. It is the coevolution of the tRNA with the aaRSs that is at the heart of the origin and evolution of the genetic code. There is also a close relationship between the accretion models of the evolving tRNA and that of the ribosome. Full article

(This article belongs to the Special Issue The Origin and Early Evolution of Life: Prebiotic Chemistry of Biomolecules)

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13 pages, 1331 KB

Open AccessArticle

A Temporal Order in 5′- and 3′- Processing of Eukaryotic tRNA^His

by Marie-Theres Pöhler, Tracy M. Roach, Heike Betat, Jane E. Jackman and Mario Mörl

Int. J. Mol. Sci. 2019, 20(6), 1384; https://doi.org/10.3390/ijms20061384 - 19 Mar 2019

Cited by 3 | Viewed by 3786

Abstract

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 3′-terminal CCA sequence that includes the site of aminoacylation, the additional 5′-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 3′-CCA and 5′-G-1 are added post-transcriptionally by tRNA nucleotidyltransferase and tRNA^His 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 tRNA^His, which might result in a temporal order to these important processing events. We show that tRNA nucleotidyltransferase accepts tRNA^His transcripts independent of the presence of G-1; however, tRNA^His 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 3′-CCA triplet and a discriminator position A73 act as positive elements for G-1 incorporation, ensuring the fidelity of G-1 addition. Full article

(This article belongs to the Special Issue Functions of Transfer RNAs 2.0)

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8 pages, 981 KB

Open AccessReview

Critical Minireview: The Fate of tRNA^Cys during Oxidative Stress in Bacillus subtilis

by Juan Campos Guillen, George H. Jones, Carlos Saldaña Gutiérrez, José Luis Hernández-Flores, Julio Alfonso Cruz Medina, José Humberto Valenzuela Soto, Sergio Pacheco Hernández, Sergio Romero Gómez and Verónica Morales Tlalpan

Biomolecules 2017, 7(1), 6; https://doi.org/10.3390/biom7010006 - 20 Jan 2017

Cited by 12 | Viewed by 6620

Abstract

Oxidative stress occurs when cells are exposed to elevated levels of reactive oxygen species that can damage biological molecules. One bacterial response to oxidative stress involves disulfide bond formation either between protein thiols or between protein thiols and low-molecular-weight (LMW) thiols. Bacillithiol was recently identified as a major low-molecular-weight thiol in Bacillus subtilis and related Firmicutes. Four genes (bshA, bshB1, bshB2, and bshC) are involved in bacillithiol biosynthesis. The bshA and bshB1 genes are part of a seven-gene operon (ypjD), which includes the essential gene cca, encoding CCA-tRNA nucleotidyltransferase. The inclusion of cca in the operon containing bacillithiol biosynthetic genes suggests that the integrity of the 3′ terminus of tRNAs may also be important in oxidative stress. The addition of the 3′ terminal CCA sequence by CCA-tRNA nucleotidyltransferase to give rise to a mature tRNA and functional molecules ready for aminoacylation plays an essential role during translation and expression of the genetic code. Any defects in these processes, such as the accumulation of shorter and defective tRNAs under oxidative stress, might exert a deleterious effect on cells. This review summarizes the physiological link between tRNA^Cys regulation and oxidative stress in Bacillus. Full article

(This article belongs to the Collection RNA Modifications)

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