RNA Nucleotidyltransferase Research Articles

Structure and mechanism of CutA, RNA nucleotidyl transferase with an unusual preference for cytosine.

Template-independent terminal ribonucleotide transferases (TENTs) catalyze the addition of nucleotide monophosphates to the 3′-end of RNA molecules regulating their fate. TENTs include poly(U) polymerases (PUPs) with a subgroup of 3′ CUCU-tagging enzymes, such as CutA in Aspergillus nidulans. CutA preferentially incorporates cytosines, processively polymerizes only adenosines and does not incorporate or extend guanosines. The basis of this peculiar specificity remains to be established. Here, we describe crystal structures of the catalytic core of CutA in complex with an incoming non-hydrolyzable CTP analog and an RNA with three adenosines, along with biochemical characterization of the enzyme. The binding of GTP or a primer with terminal guanosine is predicted to induce clashes between 2-NH2 of the guanine and protein, which would explain why CutA is unable to use these ligands as substrates. Processive adenosine polymerization likely results from the preferential binding of a primer ending with at least two adenosines. Intriguingly, we found that the affinities of CutA for the CTP and UTP are very similar and the structures did not reveal any apparent elements for specific NTP binding. Thus, the properties of CutA likely result from an interplay between several factors, which may include a conformational dynamic process of NTP recognition.

Novel biallelic TRNT1 mutations lead to atypical SIFD and multiple immune defects

Mutations in the gene encoding transfer RNA (tRNA) nucleotidyltransferase, CCA-adding 1 (TRNT1), an enzyme essential for the synthesis of the 3′-terminal CCA sequence in tRNA molecules, are associated with a rare syndrome of congenital sideroblastic anemia, B cell immunodeficiency, periodic fevers, and developmental delay (SIFD). Clinical manifestations and immunological phenotypes were assessed in a Chinese patient with novel compound heterozygous mutations in TRNT1. The patient required multiple hospitalizations starting at the age of 2 years for recurrent fevers without an infective cause. During the febrile episode, the patient was found to have microcytic hypochromic anemia, B cell lymphopenia, and hypogammaglobulinemia. Targeted gene sequencing identified novel compound heterozygous mutations in the TRNT1 gene (c.525delT, p.Leu176X; c.938T>C, p.Leu313Ser). Immunophenotyping revealed increased CD8+ T cells, CD4+ terminally differentiated effector memory helper T lymphocytes (CD4 TEMRA), and CD4+ effector memory lymphocytes (CD4 EM). Analysis of CD4+ T subsets identified decreased T follicular helper cells (Tfh) with a biased phenotype to Th2-like cells. The patient also showed a lower percentage of switched memory B (smB) cells. Additionally, defects in the cytotoxicity of the patient's NK and γδT cells were shown by CD107alpha expression. In conclusion, TRNT1 mutations may lead to multiple immune abnormality especially humoral and cytotoxicity defects, which indicate that SIFD is not only suffered ‘Predominantly antibody deficiencies’ in IUIS classification system, and further studies are needed to understand the pathogenesis of immunodeficiency in these patients.

Biochemical and structural bioinformatics studies of fungal CutA nucleotidyltransferases explain their unusual specificity toward CTP and increased tendency for cytidine incorporation at the 3'-terminal positions of synthesized tails.

Noncanonical RNA nucleotidyltransferases (NTases), including poly(A), poly(U) polymerases (PAPs/PUPs), and C/U-adding enzymes, modify 3′-ends of different transcripts affecting their functionality and stability. They contain PAP/OAS1 substrate-binding domain (SBD) with inserted NTase domain. Aspergillus nidulans CutA (AnCutA), synthesizes C/U-rich 3′-terminal extensions in vivo. Here, using high-throughput sequencing of the 3′-RACE products for tails generated by CutA proteins in vitro in the presence of all four NTPs, we show that even upon physiological ATP excess synthesized tails indeed contain an unprecedented number of cytidines interrupted by uridines and stretches of adenosines, and that the majority end with two cytidines. Strikingly, processivity assays documented that in the presence of CTP as a sole nucleotide, the enzyme terminates after adding two cytidines only. Comparison of our CutA 3D model to selected noncanonical NTases of known structures revealed substantial differences in the nucleotide recognition motif (NRM) within PAP/OAS1 SBD. We demonstrate that CutA specificity toward CTP can be partially changed to PAP or PUP by rational mutagenesis within NRM and, analogously, Cid1 PUP can be converted into a C/U-adding enzyme. Collectively, we suggest that a short cluster of amino acids within NRM is a determinant of NTases’ substrate preference, which may allow us to predict their specificity.

TRNT1 deficiency: clinical, biochemical and molecular genetic features.

BackgroundTRNT1 (CCA-adding transfer RNA nucleotidyl transferase) enzyme deficiency is a new metabolic disease caused by defective post-transcriptional modification of mitochondrial and cytosolic transfer RNAs (tRNAs).ResultsWe investigated four patients from two families with infantile-onset cyclical, aseptic febrile episodes with vomiting and diarrhoea, global electrolyte imbalance during these episodes, sideroblastic anaemia, B lymphocyte immunodeficiency, retinitis pigmentosa, hepatosplenomegaly, exocrine pancreatic insufficiency and renal tubulopathy. Other clinical features found in children include sensorineural deafness, cerebellar atrophy, brittle hair, partial villous atrophy and nephrocalcinosis.Whole exome sequencing and bioinformatic filtering were utilised to identify recessive compound heterozygous TRNT1 mutations (missense mutation c.668T>C, p.Ile223Thr and a novel splice mutation c.342+5G>T) segregating with disease in the first family. The second family was found to have a homozygous TRNT1 mutation (c.569G>T), p.Arg190Ile, (previously published).We found normal mitochondrial translation products using passage matched controls and functional perturbation of 3’ CCA addition to mitochondrial tRNAs (tRNACys, tRNALeuUUR and tRNAHis) in fibroblasts from two patients, demonstrating a pathomechanism affecting the CCA addition to mt-tRNAs. Acute management of these patients included transfusion for anaemia, fluid and electrolyte replacement and immunoglobulin therapy. We also describe three-year follow-up findings after treatment by bone marrow transplantation in one patient, with resolution of fever and reversal of the abnormal metabolic profile.ConclusionsOur report highlights that TRNT1 mutations cause a spectrum of disease ranging from a childhood-onset complex disease with manifestations in most organs to an adult-onset isolated retinitis pigmentosa presentation. Systematic review of all TRNT1 cases and mutations reported to date revealed a distinctive phenotypic spectrum and metabolic and other investigative findings, which will facilitate rapid clinical recognition of future cases.

Compartmentalization of destabilized enzyme-mRNA-ribosome complexes generated by ribosome display: a novel tool for the directed evolution of enzymes

We have developed an in vitro evolution method for the selection for catalytic activity under the conditions of free intermolecular interaction between the enzyme and a substrate. The destabilized ternary enzyme-mRNA-ribosome complexes generated by a ribosome display of the mutant library are compartmentalized in vitro by forming a water-in-oil emulsion in such a way, that every droplet would on average contain no more than a single complex. After the complex dissociates within the droplet, the released enzyme molecule is free to interact with a substrate under the selection pressure on all its enzymatic properties (substrate binding, product formation, rate acceleration and turnover) simultaneously-an opportunity for the most efficient selection for catalytic activity. By using the M-MuLV reverse transcriptase as a model, we demonstrated the high efficiency of the method selecting for mutants synthesizing cDNA at increased temperature. A slightly modified compartmentalized ribosome display (CRD) could be used for the selection of other enzymes activities (e.g. DNA polymerase, RNA or DNA ligase terminal nucleotidyl transferase activity). Employment of microfluidics technique could broaden the scope of CRD technique furthermore providing an opportunity to select almost any enzyme at single molecule level under desired conditions.

Geobacter sulfurreducens Contains Separate C- and A-Adding tRNA Nucleotidyltransferases and a Poly(A) Polymerase

The genome of Geobacter sulfurreducens contains three genes whose sequences are quite similar to sequences encoding known members of an RNA nucleotidyltransferase superfamily that includes tRNA nucleotidyltransferases and poly(A) polymerases. Reverse transcription-PCR using G. sulfurreducens total RNA demonstrated that the genes encoding these three proteins are transcribed. These genes, encoding proteins designated NTSFI, NTSFII, and NTSFIII, were cloned and overexpressed in Escherichia coli. The corresponding enzymes were purified and assayed biochemically, resulting in identification of NTSFI as a poly(A) polymerase, NTSFII as a C-adding tRNA nucleotidyltransferase, and NTSFIII as an A-adding tRNA nucleotidyltransferase. Analysis of G. sulfurreducens rRNAs and mRNAs revealed the presence of heteropolymeric RNA 3' tails. This is the first characterization of a bacterial system that expresses separate C- and A-adding tRNA nucleotidyltransferases and a poly(A) polymerase.

Special issue: Novel RNA nucleotidyl transferases and gene regulation

Special issue: Novel RNA nucleotidyl transferases and gene regulation

UTP-bound and Apo Structures of a Minimal RNA Uridylyltransferase

3′-Uridylylation of RNA is emerging as a phylogenetically widespread phenomenon involved in processing events as diverse as uridine insertion/deletion RNA editing in mitochondria of trypanosomes and small nuclear RNA (snRNA) maturation in humans. This reaction is catalyzed by terminal uridylyltransferases (TUTases), which are template-independent RNA nucleotidyltransferases that specifically recognize UTP and belong to a large enzyme superfamily typified by DNA polymerase β. Multiple TUTases, recently identified in trypanosomes, as well as a U6 snRNA-specific TUTase enzyme in humans, are highly divergent at the protein sequence level. However, they all possess conserved catalytic and UTP recognition domains, often accompanied by various auxiliary modules present at the termini or between conserved domains. Here we report identification, structural and biochemical analyses of a novel trypanosomal TUTase, TbTUT4, which represents a minimal catalytically active RNA uridylyltransferase. The TbTUT4 consists of only two domains that define the catalytic center at the bottom of the nucleoside triphosphate and RNA substrate binding cleft. The 2.0 Å crystal structure reveals two significantly different conformations of this TUTase: one molecule is in a relatively open apo conformation, whereas the other displays a more compact TUTase–UTP complex. A single nucleoside triphosphate is bound in the active site by a complex network of interactions between amino acid residues, a magnesium ion and highly ordered water molecules with the UTP's base, ribose and phosphate moieties. The structure-guided mutagenesis and cross-linking studies define the amino acids essential for catalysis, uracil base recognition, ribose binding and phosphate coordination by uridylyltransferases. In addition, the cluster of positively charged residues involved in RNA binding is identified. We also report a 2.4 Å crystal structure of TbTUT4 with the bound 2′ deoxyribonucleoside, which provides the structural basis of the enzyme's preference toward ribonucleotides.

A Phylogeny of Bacterial RNA Nucleotidyltransferases: Bacillus halodurans Contains Two tRNA Nucleotidyltransferases

We have analyzed the distribution of RNA nucleotidyltransferases from the family that includes poly(A) polymerases (PAP) and tRNA nucleotidyltransferases (TNT) in 43 bacterial species. Genes of several bacterial species encode only one member of the nucleotidyltransferase superfamily (NTSF), and if that protein functions as a TNT, those organisms may not contain a poly(A) polymerase I like that of Escherichia coli. The genomes of several of the species examined encode more than one member of the nucleotidyltransferase superfamily. The function of some of those proteins is known, but in most cases no biochemical activity has been assigned to the NTSF. The NTSF protein sequences were used to construct an unrooted phylogenetic tree. To learn more about the function of the NTSFs in species whose genomes encode more than one, we have examined Bacillus halodurans. We have demonstrated that B. halodurans adds poly(A) tails to the 3' ends of RNAs in vivo. We have shown that the genes for both of the NTSFs encoded by the B. halodurans genome are transcribed in vivo. We have cloned, overexpressed, and purified the two NTSFs and have shown that neither functions as poly(A) polymerase in vitro. Rather, the two proteins function as tRNA nucleotidyltransferases, and our data suggest that, like some of the deep branching bacterial species previously studied by others, B. halodurans possesses separate CC- and A-adding tRNA nucleotidyltransferases. These observations raise the interesting question of the identity of the enzyme responsible for RNA polyadenylation in Bacillus.

Mechanism of transfer RNA maturation by CCA-adding enzyme without using an oligonucleotide template.

Transfer RNA nucleotidyltransferases (CCA-adding enzymes) are responsible for the maturation or repair of the functional 3' end of tRNAs by means of the addition of the essential nucleotides CCA. However, it is unclear how tRNA nucleotidyltransferases polymerize CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template. Here we describe the crystal structure of the Archaeoglobus fulgidus tRNA nucleotidyltransferase in complex with tRNA. We also present ternary complexes of this enzyme with both RNA duplex mimics of the tRNA acceptor stem that terminate with the nucleotides C74 or C75, as well as the appropriate incoming nucleoside 5'-triphosphates. A single nucleotide-binding pocket exists whose specificity for both CTP and ATP is determined by the protein side chain of Arg 224 and backbone phosphates of the tRNA, which are non-complementary to and thus exclude UTP and GTP. Discrimination between CTP or ATP at a given addition step and at termination arises from changes in the size and shape of the nucleotide binding site that is progressively altered by the elongating 3' end of the tRNA.

Parasites as probes for biodiversity : S. L. Gardner & M. L. Campbell, Journal of Parasitology, 78(4), 1992, pp 596–600

The synthesis of RNA catalyzed by RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) is inhibited by several anionic dyes; the most effective being congo red. In contrast to other inhibitors of RNA polymerase which act by binding to the template (i.e. actinomycin D, proflavin), congo red appears to inhibit by binding to the enzyme, and the extent of inhibition is independent of the nature of the primer. The enzyme can be protected against the action of the dye by adding albumin which will bind the dye, or by increasing the amount of enzyme in order to decrease the dye to enzyme ratio.

Images of evolution: origin of spontaneous RNA replication waves.

Self-replicating molecules set up traveling concentration waves that propagate in an aqueous enzyme solution. The velocity of each wave provides an accurate (+/- 0.1%) noninvasive measure of fitness for the RNA species currently growing in its front. Evolution may be followed from changes in the front velocity, and these differ from wave to wave. Thousands of controlled evolution reactions in traveling waves have been monitored in parallel to obtain quantitative images of the stochastic process of natural selection. An RNA polymerase (RNA-dependent RNA nucleotidyltransferase, EC 2.7.7.6), extracted from bacteria infected by the Q beta RNA virus, catalyzes the replication. The traveling waves that arise spontaneously without added RNA provide a model system for major evolutionary change.

Cyanobacterial RNA polymerase genes rpoC1 and rpoC2 correspond to rpoC of Escherichia coli

The DNA-dependent RNA polymerase (ribonucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) of cyanobacteria contains a unique core component, gamma, which is absent from the RNA polymerases of other eubacteria (G. J. Schneider, N. E. Tumer, C. Richaud, G. Borbely, and R. Haselkorn, J. Biol. Chem. 262:14633-14639, 1987). We present the complete nucleotide sequence of rpoC1, the gene encoding the gamma subunit, from the heterocystous cyanobacterium Nostoc commune UTEX 584. The derived amino acid sequence of gamma (621 residues) corresponds with the amino-terminal portion of the beta' polypeptide of Escherichia coli RNA polymerase. A second gene in N. commune UTEX 584, rpoC2, encodes a protein which shows correspondence with the carboxy-terminal portion of the E. coli beta' subunit. The rpoBC1C2 genes of N. commune UTEX 584 are present in single copies and are arranged in the order rpoBC1C2, and the coding regions are separated by short AT-rich spacer regions which have the potential to form very stable secondary structures. Our data indicate the occurrence of divergent evolution of structure in the eubacterial DNA-dependent RNA polymerase.

Red light irradiation ofWolffia arrhiza does not influence transcription activity of isolated DNA-dependent RNA polymerase II

DNA-dependent RNA polymerase II (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6.) from duckweed Wolffia arrhiza (L.) HORKEL ex WIMMER was partially purified by polyethylenimine precipitation and DEAE cellulose ion exchange chromatography. Whole plants were irradiated with red light pulses. After a dark period of 2 to 12 h there is no effect of light pretreatment on the total or specific activity of the purified enzyme.

Transfer RNA nucleotidyl transferase from Artemia. Developmental changes and integrity of transfer RNA

1. 1. Encysted gastrulae of Artemia contain a transfer RNA nucleotidyl transferase activity which incorporates AMP from ATP into 3′ incomplete tRNA chains. 2. 2. The activity is maximally active with Mg 2+, uses tRNA as acceptor substrate more efficiently than poly (A) and the reaction product is sensitive to pancreatic ribonuclease. These characteristics are different from those described for the poly (A) polymerase, an enzyme also present in Artemia which incorporates AMP residues into RNA chains. 3. 3. The transferase activity increases (four fold) during early larval development reaching a maximum in larvae of 30 hr. The low levels of transferase activity in the encysted gastrulae is not paralleled by lack in the integrity of the amino acid acceptor end of the tRNA chains. This terminal end of tRNA molecules is not impaired along the first 50 hours of Artemia development.

Some properties of DNA-dependent RNA polymerase from Streptomyces granaticolor.

RNA nucleotidyltransferase from Streptomyces granaticolor was purified and some of its properties were investigated. The temperature optimum of the enzyme is 35 degrees C, pH optimum 7.8-8.4. Antibodies against the beta subunit of the enzyme from Bacillus subtilis immunologically cross-react with the beta subunit of the enzyme from S. granaticolor. Antibodies against the beta' subunit of the enzyme from B. subtilis immunologically cross-react with both beta and beta' subunits of the enzyme from S. granaticolor.

Effect of phenobarbital on the activity of RNA polymerase II during diethylnitrosamine-induced hepatocarcinogenesis in the rat

The effects of dietary phenobarbital (PB) on RNA polymerase II (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2. 7. 7. 6) in the liver of male Sprague-Dawley rats previously administered diethylnitrosamine (DENA) were studied. When a diet containing 0.05% phenobarbital was administered, the activity of DNA-dependent RNA polymerase II in the liver nuclei of rats was temporarily increased 1 and 2 weeks after the onset of feeding and expressed to be relatively higher afterwards, compared with the activity in rats fed the control diets.

Wheat-embryo ribonucleates. XV. Characterization of a fraction of RNA subject to conspicuous terminal labeling during early germination of wheat embryos

Various techniques have been used to characterize the rapidly migrating electrophoretic components (RMEC) in an RNA fraction which is subject to conspicuous terminal labeling (mostly adenosine) when wheat embryos are pulse labeled with 3H-labeled nucleosides during early germination. Detection of terminally labeled RMEC RNA is most favoured during early germination when labeling of 3′-hydroxyl termini in preexisting RNA, catalyzed by RNA nucleotidyltransferases, is greatest in relation to nonterminal labeling of nascent RNA, catalyzed by RNA polymerases. The RMEC RNA is tenaciously associated with a fraction of high-molecular-weight RNA and it can be subdivided and classified into two fractions, RMEC-1 and RMEC-2, by electrophoresis in polyacrylamide, gel filtration through Sephadex, or assay of amino acid acceptance. The RMEC-1 RNA and bulk wheat-embryo tRNA have similar capacities to accept a wide variety of amino acids, but RMEC-2 RNA does not accept any of the amino acids tested. RMEC-2 RNA is broadly heterodisperse and one of its component polynucleotides has been identified by sequence analysis as a tridecanucleotide fragment from the 3′-hydroxyl end of 5.8S rRNA. This study was undertaken as part of a broader program in which isotopic labeling and analysis of nucleates and proteins in germinating wheat embryos have been used to detect signal events and to evaluate their physiological significance. It is concluded that conspicuous terminal labeling of RMEC RNA during early germination of wheat embryos is unlikely to be of physiological importance. However, isotopic labeling and analysis of the nucleates and proteins in germinating embryos has uncovered other signal events which appear to be of physiological importance. Discovery of the existence of a number of developmentally regulated proteins and the way in which discovery of these proteins is expected to direct the course of future investigations in the laboratory are subjects of a brief discussion.

The quaternary structure of Escherichia coli RNA polymerase studied with (scanning) transmission (immuno)electron microscopy.

A model for the quaternary structure of Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) is presented. It is based on results from classification of profiles of enzyme molecules, and from application of immuno electron microscopy. Classification of molecules, prepared with the single carbon layer technique, was first achieved for images recorded in dark field with the scanning transmission electron microscope and later on for images recorded in bright-field transmission electron microscopy. It results in five approximately equally sized groups, containing about 80% of the core enzyme profiles. Holoenzyme profiles can be grouped into the same classes, and have approximately the same dimensions (9 nm X 16 nm). Based on the shapes and sizes of the classified profiles, a tentative model for core enzyme has been constructed. Correlation of shadow projections of this model, with the distributions of attachment sites of antibodies against alpha, beta, beta' and sigma over the profiles, has led to models for core and holoenzyme in which the subunits are localized. The model is compared with literature data on the quaternary structure of RNA polymerase.

Purification of DNA-dependent RNA polymerase from Streptomyces granaticolor.

RNA nucleotidyltransferase (EC 2.7.7.6) of Streptomyces granaticolor was purified by precipitation with polymin P and ammonium sulphate, affinity chromatography on DNA- cellulose and gell filtration on Biogel A 1.5 m. SDS-polyacrylamide gel electrophoresis revealed 8 protein bands of molar mass ranging from 37 to 130 kg/mol. Proteins of molar mass of 130 and 120 kg/mol were identified to be beta and beta subunits, respectively. The role of other subunits of the enzyme is discussed.