Evolution of tRNA nucleotidyltransferases: a small deletion generated CC-adding enzymes.
ABSTRACT: CCA-adding enzymes are specialized polymerases that add a specific sequence (C-C-A) to tRNA 3' ends without requiring a nucleic acid template. In some organisms, CCA synthesis is accomplished by the collaboration of evolutionary closely related enzymes with partial activities (CC and A addition). These enzymes carry all known motifs of the catalytic core found in CCA-adding enzymes. Therefore, it is a mystery why these polymerases are restricted in their activity and do not synthesize a complete CCA terminus. Here, a region located outside of the conserved motifs was identified that is missing in CC-adding enzymes. When recombinantly introduced from a CCA-adding enzyme, the region restores full CCA-adding activity in the resulting chimera. Correspondingly, deleting the region in a CCA-adding enzyme abolishes the A-incorporating activity, also leading to CC addition. The presence of the deletion was used to predict the CC-adding activity of putative bacterial tRNA nucleotidyltransferases. Indeed, two such enzymes were experimentally identified as CC-adding enzymes, indicating that the existence of the deletion is a hallmark for this activity. Furthermore, phylogenetic analysis of identified and putative CC-adding enzymes indicates that this type of tRNA nucleotidyltransferases emerged several times during evolution. Obviously, these enzymes descend from CCA-adding enzymes, where the occurrence of the deletion led to the restricted activity of CC addition. A-adding enzymes, however, seem to represent a monophyletic group that might also be ancestral to CCA-adding enzymes. Yet, experimental data indicate that it is possible that A-adding activities also evolved from CCA-adding enzymes by the occurrence of individual point mutations.
Project description:For efficient aminoacylation, tRNAs carry the conserved 3'-terminal sequence C-C-A, which is synthesized by highly specific tRNA nucleotidyltransferases (CCA-adding enzymes). In several prokaryotes, this function is accomplished by separate enzymes for CC- and A-addition. As A-adding enzymes carry an N-terminal catalytic core identical to that of CCA-adding enzymes, it is unclear why their activity is restricted. Here, it is shown that C-terminal deletion variants of A-adding enzymes acquire full and precise CCA-incorporating activity. The deleted region seems to be responsible for tRNA primer selection, restricting the enzyme's specificity to tRNAs ending with CC. The data suggest that A-adding enzymes carry an intrinsic CCA-adding activity that can be reactivated by the introduction of deletions in the C-terminal domain. Furthermore, a unique subtype of CCA-adding enzymes could be identified that evolved out of A-adding enzymes, suggesting that mutations and deletions in nucleotidyltransferases can lead to altered and even more complex activities, as a simple A-incorporation is converted into sequence-specific addition of C and A residues. Such activity-modifying events may have had an important role in the evolution of tRNA nucleotidyltransferases.
Project description: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.
Project description:CCA-adding enzymes are highly specific RNA polymerases that synthesize and maintain the sequence CCA at the tRNA 3‘-end. Here, we investigated the impact of cold adaptation on the reactivity and specificity of CCA-adding enzymes from psychrophilic bacteria. A comparative study of the corresponding enzymes from closely related psychro-, meso-, and thermophilic Bacillales indicates that the cold-adapted enzymes show a considerable error rate during CCA synthesis, resulting in additional incorporations of C and A residues. It seems that the activity of psychrophilic CCA-adding enzymes is not only achieved at the expense of structural stability, reaction velocity and substrate affinity, but also results in a reduced polymerization fidelity. Overall design: in vivo tRNA from four Bacillales species was isolated from exponential and stationary growth phase each in triplicates resulting in a total of 24 samples. Sequencing libraries were created for small RNA species and sequenced on 3 runs of a MiSeq using 8 indices. Reads were analyzed regarding 3'-ends of tRNA and results summed up as a mean for the 8 conditions investigated. Results from B. subtilis were regarded as reference sample for a mesophilic Bacillales species. Differences towards psychophilic Planococcus halocryophilus and Exiguobacterium sibiricum and thermophilic Geobacills stearothermophilus were investigated.
Project description: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.
Project description:Cold adaptation is an evolutionary process that has dramatic impact on enzymatic activity. Increased flexibility of the protein structure represents the main evolutionary strategy for efficient catalysis and reaction rates in the cold, but is achieved at the expense of structural stability. This results in a significant activity-stability tradeoff, as it was observed for several metabolic enzymes. In polymerases, however, not only reaction rates, but also fidelity plays an important role, as these enzymes have to synthesize copies of DNA and RNA as exact as possible. Here, we investigate the effects of cold adaptation on the highly accurate CCA-adding enzyme, an RNA polymerase that uses an internal amino acid motif within the flexible catalytic core as a template to synthesize the CCA triplet at tRNA 3'-ends. As the relative orientation of these residues determines nucleotide selection, we characterized how cold adaptation impacts template reading and fidelity. In a comparative analysis of closely related psychro-, meso-, and thermophilic enzymes, the cold-adapted polymerase shows a remarkable error rate during CCA synthesis in vitro as well as in vivo. Accordingly, CCA-adding activity at low temperatures is not only achieved at the expense of structural stability, but also results in a reduced polymerization fidelity.
Project description:Transfer RNAs (tRNAs) require the absolutely conserved sequence motif CCA at their 3'-ends, representing the site of aminoacylation. In the majority of organisms, this trinucleotide sequence is not encoded in the genome and thus has to be added post-transcriptionally by the CCA-adding enzyme, a specialized nucleotidyltransferase. In eukaryotic genomes this ubiquitous and highly conserved enzyme family is usually represented by a single gene copy. Analysis of published sequence data allows us to pin down the unusual evolution of eukaryotic CCA-adding enzymes. We show that the CCA-adding enzymes of animals originated from a horizontal gene transfer event in the stem lineage of Holozoa, i.e. Metazoa (animals) and their unicellular relatives, the Choanozoa. The tRNA nucleotidyltransferase, acquired from an ?-proteobacterium, replaced the ancestral enzyme in Metazoa. However, in Choanoflagellata, the group of Choanozoa that is closest to Metazoa, both the ancestral and the horizontally transferred CCA-adding enzymes have survived. Furthermore, our data refute a mitochondrial origin of the animal tRNA nucleotidyltransferases.
Project description:CCA-adding enzymes build and repair the 3'-terminal CCA sequence of tRNA. These unusual RNA polymerases use either a ribonucleoprotein template (class I) or pure protein template (class II) to form mock base pairs with the Watson-Crick edges of incoming CTP and ATP. Guided by the class II Bacillus stearothermophilus CCA-adding enzyme structure, we introduced mutations designed to reverse the polarity of hydrogen bonds between the nucleobases and protein template. We were able to transform the CCA-adding enzyme into a (U,G)-adding enzyme that incorporates UTP and GTP instead of CTP and ATP; we transformed the related Aquifex aeolicus CC- and A-adding enzymes into UU- and G-adding enzymes and Escherichia coli poly(A) polymerase into a poly(G) polymerase; and we transformed the B. stearothermophilus CCA-adding enzyme into a poly(C,A) polymerase by mutations in helix J that appear, based on the apoenzyme structure, to sterically limit addition to CCA. We also transformed the B. stearothermophilus CCA-adding enzyme into a dCdCdA-adding enzyme by mutating an arginine that interacts with the incoming ribose 2' hydroxyl. Most importantly, we found that mutations in helix J can affect the specificity of the nucleotide binding site some 20 A away, suggesting that the specificity of both class I and II enzymes may be dictated by an intricate network of hydrogen bonds involving the protein, incoming nucleotide, and 3' end of the tRNA. Collaboration between RNA and protein in the form of a ribonucleoprotein template may help to explain the evolutionary diversity of the nucleotidyltransferase family.
Project description:CCA-adding enzymes are highly specific RNA polymerases that synthesize and maintain the sequence CCA at the tRNA 3'-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'-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.
Project description:Correct synthesis and maintenance of functional tRNA 3'-CCA-ends is a crucial prerequisite for aminoacylation and must be achieved by the phylogenetically diverse group of tRNA nucleotidyltransferases. While numerous reports on the in vitro characterization exist, robust analysis under in vivo conditions is lacking. Here, we utilize Escherichia coli RNase T, a tRNA-processing enzyme responsible for the tRNA-CCA-end turnover, to generate an in vivo system for the evaluation of A-adding activity. Expression of RNase T results in a prominent growth phenotype that renders the presence of a CCA- or A-adding enzyme essential for cell survival in an E. coli ?cca background. The distinct growth fitness allows for both complementation and selection of enzyme variants in a natural environment. We demonstrate the potential of our system via detection of altered catalytic efficiency and temperature sensitivity. Furthermore, we select functional enzyme variants out of a sequence pool carrying a randomized codon for a highly conserved position essential for catalysis. The presented E. coli-based approach opens up a wide field of future studies including the investigation of tRNA nucleotidyltransferases from all domains of life and the biological relevance of in vitro data concerning their functionality and mode of operation.
Project description:CCA-adding enzymes [ATP(CTP):tRNA nucleotidyltransferases] add CCA onto the 3' end of transfer RNA (tRNA) precursors without using a nucleic acid template. Although the mechanism by which cytosine (C) is selected at position 75 of tRNA has been established, the mechanism by which adenine (A) is selected at position 76 remains elusive. Here, we report five cocrystal structures of the enzyme complexed with both a tRNA mimic and nucleoside triphosphates under catalytically active conditions. These structures suggest that adenosine 5'-monophosphate is incorporated onto the A76 position of the tRNA via a carboxylate-assisted, one-metal-ion mechanism with aspartate 110 functioning as a general base. The discrimination against incorporation of cytidine 5'-triphosphate (CTP) at position 76 arises from improper placement of the ? phosphate of the incoming CTP, which results from the interaction of C with arginine 224 and prevents the nucleophilic attack by the 3' hydroxyl group of cytidine75.