Dual expression of CCA-adding enzyme and RNase T in Escherichia coli generates a distinct cca growth phenotype with diverse applications.
ABSTRACT: 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: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: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: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:tRNA maturation and quality control are crucial for proper functioning of these transcripts in translation. In several organisms, defective tRNAs were shown to be tagged by poly(A) or CCACCA tails and subsequently degraded by 3'-exonucleases. In a deep-sequencing analysis of tRNA 3'-ends, we detected the CCACCA tag also in Escherichia coli However, this tag closely resembles several 3'-trailers of tRNA precursors targeted for maturation and not for degradation. Here, we investigate the ability of two important exonucleases, RNase R and RNase T, to distinguish tRNA precursors with a native 3'-trailer from tRNAs with a CCACCA tag. Our results show that the degrading enzyme RNase R breaks down both tRNAs primed for degradation as well as precursor transcripts, indicating that it is a rather nonspecific RNase. RNase T, a main processing exonuclease involved in trimming of 3'-trailers, is very inefficient in converting the CCACCA-tagged tRNA into a mature transcript. Hence, while both RNases compete for trailer-containing tRNA precursors, the inability of RNase T to process CCACCA tails ensures that defective tRNAs cannot reenter the functional tRNA pool, representing a safeguard to avoid detrimental effects of tRNAs with erroneous integrity on protein synthesis. Furthermore, these data indicate that the RNase T-mediated end turnover of the CCA sequence represents a means to deliver a tRNA to a repeated quality control performed by the CCA-adding enzyme. Hence, originally described as a futile side reaction, the tRNA end turnover seems to fulfill an important function in the maintenance of the tRNA pool in the cell.
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.
Project description: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: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:Post-transcriptional non-template additions of nucleotides to 3'-ends of RNAs play important roles in the stability and function of RNA molecules. Although tRNA nucleotidyltransferase (CCA-adding enzyme) is known to add CCA trinucleotides to 3'-ends of tRNAs, whether other RNA species can be endogenous substrates of CCA-adding enzyme has not been widely explored yet. Herein, we used YAMAT-seq to identify non-tRNA substrates of CCA-adding enzyme. YAMAT-seq captures RNA species that form secondary structures with 4-nt protruding 3'-ends of the sequence 5'-NCCA-3', which is the hallmark structure of RNAs that are generated by CCA-adding enzyme. By executing YAMAT-seq for human breast cancer cells and mining the sequence data, we identified novel candidate substrates of CCA-adding enzyme. These included fourteen 'CCA-RNAs' that only contain CCA as non-genomic sequences, and eleven 'NCCA-RNAs' that contain CCA and other nucleotides as non-genomic sequences. All newly-identified (N)CCA-RNAs were derived from the mitochondrial genome and were localized in mitochondria. Knockdown of CCA-adding enzyme severely reduced the expression levels of (N)CCA-RNAs, suggesting that the CCA-adding enzyme-catalyzed CCA additions stabilize the expression of (N)CCA-RNAs. Furthermore, expression levels of (N)CCA-RNAs were severely reduced by various cellular treatments, including UV irradiation, amino acid starvation, inhibition of mitochondrial respiratory complexes, and inhibition of the cell cycle. These results revealed a novel CCA-mediated regulatory pathway for the expression of mitochondrial non-coding RNAs.
Project description:CCA-adding enzymes synthesize and maintain the C-C-A sequence at the tRNA 3'-end, generating the attachment site for amino acids. While tRNAs are the most prominent substrates for this polymerase, CCA additions on non-tRNA transcripts are described as well. To identify general features for substrate requirement, a pool of randomized transcripts was incubated with the human CCA-adding enzyme. Most of the RNAs accepted for CCA addition carry an acceptor stem-like terminal structure, consistent with tRNA as the main substrate group for this enzyme. While these RNAs show no sequence conservation, the position upstream of the CCA end was in most cases represented by an adenosine residue. In tRNA, this position is described as discriminator base, an important identity element for correct aminoacylation. Mutational analysis of the impact of the discriminator identity on CCA addition revealed that purine bases (with a preference for adenosine) are strongly favoured over pyrimidines. Furthermore, depending on the tRNA context, a cytosine discriminator can cause a dramatic number of misincorporations during CCA addition. The data correlate with a high frequency of adenosine residues at the discriminator position observed in vivo. Originally identified as a prominent identity element for aminoacylation, this position represents a likewise important element for efficient and accurate CCA addition.
Project description:CCA-adding enzyme builds the 3'-end CCA of tRNA without a nucleic acid template. The mechanism for the maintenance of fidelity during the CCA-adding reaction remains elusive. Here, we present almost a dozen complex structures of the class I CCA-adding enzyme and tRNA mini-helices (mini-D(73)N(74), mini-D(73)N(74)C(75) and mini-D(73)C(74)N(75); D(73) is a discriminator nucleotide and N is either A, G, or U). The mini-D(73)N(74) complexes adopt catalytically inactive open forms, and CTP shifts the enzymes to the active closed forms and allows N(74) to flip for CMP incorporation. In contrast, unlike the catalytically active closed form of the mini-D(73)C(74)C(75) complex, the mini-D(73)N(74)C(75) and mini-D(73)C(74)N(75) complexes adopt inactive open forms. Only the mini-D(73)C(74)U(75) accepts AMP to a similar extent as mini-D(73)C(74)C(75), and ATP shifts the enzyme to a closed, active form and allows U(75) to flip for AMP incorporation. These findings suggest that the 3'-region of RNA is proofread, after two nucleotide additions, in the closed, active form of the complex at the AMP incorporation stage. This proofreading is a prerequisite for the maintenance of fidelity for complete CCA synthesis.