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At least 19 recordsLinked to original sources
PubMed2005

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.

Amino Acid Sequence↗
PubMed1990

Isolation of a temperature-sensitive mutant with an altered tRNA nucleotidyltransferase and cloning of the gene encoding tRNA nucleotidyltransferase in the yeast Saccharomyces cerevisiae.

We have isolated a yeast mutant, ts352, that is temperature-sensitive for growth. The mutation has a general effect on mRNA metabolism and a specific effect on tRNA biosynthesis. Cells shifted to the nonpermissive temperature accumulate tRNAs that are shorter than mature tRNAs. The increased ability of these tRNAs to accept ATP demonstrates that growth of the ts352 mutant at the nonpermissive temperature results in accumulation of tRNA with defective 3' ends. The activity of ATP (CTP):tRNA-specific tRNA nucleotidyltransferase can readily be measured in extracts from wild type but not mutant cells. We have cloned and sequenced the wild type allele of the ts352 gene and find significant similarity between the yeast protein sequence predicted from the DNA sequence and the protein predicted from the sequence of the Escherichi coli tRNA nucleotidyltransferase gene. Expression of the yeast gene on a multicopy plasmid increases the activity of the tRNA nucleotidyltransferase in extracts. We conclude that the defect in the ts352 mutant is in the gene coding for yeast tRNA nucleotidyltransferase and that we have isolated the yeast gene that codes for this enzyme.

Amino Acid Sequence↗
PubMed2004

The HD domain of the Escherichia coli tRNA nucleotidyltransferase has 2',3'-cyclic phosphodiesterase, 2'-nucleotidase, and phosphatase activities.

In all mature tRNAs, the 3'-terminal CCA sequence is synthesized or repaired by a template-independent nucleotidyltransferase (ATP(CTP):tRNA nucleotidyltransferase; EC 2.7.7.25). The Escherichia coli enzyme comprises two domains: an N-terminal domain containing the nucleotidyltransferase activity and an uncharacterized C-terminal HD domain. The HD motif defines a superfamily of metal-dependent phosphohydrolases that includes a variety of uncharacterized proteins and domains associated with nucleotidyltransferases and helicases from bacteria, archaea, and eukaryotes. The C-terminal HD domain in E. coli tRNA nucleotidyltransferase demonstrated Ni(2+)-dependent phosphatase activity toward pyrophosphate, canonical 5'-nucleoside tri- and diphosphates, NADP, and 2'-AMP. Assays with phosphodiesterase substrates revealed surprising metal-independent phosphodiesterase activity toward 2',3'-cAMP, -cGMP, and -cCMP. Without metal or in the presence of Mg(2+), the tRNA nucleotidyltransferase hydrolyzed 2',3'-cyclic substrates with the formation of 2'-nucleotides, whereas in the presence of Ni(2+), the protein also produced some 3'-nucleotides. Mutations at the conserved His-255 and Asp-256 residues comprising the C-terminal HD domain of this protein inactivated both phosphodiesterase and phosphatase activities, indicating that these activities are associated with the HD domain. Low concentrations of the E. coli tRNA (10 nm) had a strong inhibiting effect on both phosphatase and phosphodiesterase activities. The competitive character of inhibition by tRNA suggests that it might be a natural substrate for these activities. This inhibition was completely abolished by the addition of Mg(2+), Mn(2+), or Ca(2+), but not Ni(2+). The data suggest that the phosphohydrolase activities of the HD domain of the E. coli tRNA nucleotidyltransferase are involved in the repair of the 3'-CCA end of tRNA.

Adenosine Monophosphate↗
PubMed1999

DNA polymerase beta-like nucleotidyltransferase superfamily: identification of three new families, classification and evolutionary history.

A detailed analysis of the polbeta superfamily of nucleotidyltransferases was performed using computer methods for iterative database search, multiple alignment, motif analysis and structural modeling. Three previously uncharacterized families of predicted nucleotidyltransferases are described. One of these new families includes small proteins found in all archaea and some bacteria that appear to consist of the minimal nucleotidyltransferase domain and may resemble the ancestral state of this superfamily. Another new family that is specifically related to eukaryotic polyA polymerases is typified by yeast Trf4p and Trf5p proteins that are involved in chromatin remodeling. The TRF family is represented by multiple members in all eukaryotes and may be involved in yet unknown nucleotide polymerization reactions required for maintenance of chromatin structure. Another new family of bacterial and archaeal nucleotidyltransferases is predicted to function in signal transduction since, in addition to the nucleotidyltransferase domain, these proteins contain ligand-binding domains. It is further shown that the catalytic domain of gamma proteobacterial adenylyl cyclases is homologous to the polbeta superfamily nucleotidyltransferases which emphasizes the general trend for the origin of signal-transducing enzymes from those involved in replication, repair and RNA processing. Classification of the polbeta superfamily into distinct families and examination of their phyletic distribution suggests that the evolution of this type of nucleotidyltransferases may have included bursts of rapid divergence linked to the emergence of new functions as well as a number of horizontal gene transfer events.

Adenylyl Cyclases↗
PubMed1997

Functional overlap of tRNA nucleotidyltransferase, poly(A) polymerase I, and polynucleotide phosphorylase.

Repair of the 3'-terminal -CCA sequence of tRNA generally requires the action of the enzyme tRNA nucleotidyltransferase. However, in Escherichia coli in the absence of this enzyme, a decreased level of tRNA end repair continues. To ascertain the enzymes responsible for this residual repair, mutant strains were constructed lacking tRNA nucleotidyltransferase and other enzymes potentially involved in the process, poly(A) polymerase I and polynucleotide phosphorylase (PNPase). Strains lacking tRNA nucleotidyltransferase and either one of the other enzymes displayed decreased growth rates and increased levels of defective tRNA compared with the single cca mutant. Triple mutants lacking all three enzymes grew very slowly, had even more defective tRNA, and were devoid of activity incorporating AMP into tRNA-C-C. Overexpression of poly(A) polymerase I, but not PNPase, partially compensated for the absence of tRNA nucleotidyltransferase. These data show that poly(A) polymerase I and PNPase participate in the end repair process and are required to maintain functional tRNA levels when tRNA nucleotidyltransferase is absent.

Adenosine Monophosphate↗
PubMed2001

Regiospecificity assignment for the reaction of kanamycin nucleotidyltransferase from Staphylococcus aureus.

Aminoglycoside nucleotidyltransferases catalyze the transfer of a nucleoside monophosphoryl group from a nucleotide to a hydroxyl group of an aminoglycoside antibiotic. Kanamycin nucleotidyltransferase [ANT (4',4' ')-I] from Staphylococcus aureus confers resistance to numerous aminoglycosides with a 4' or 4' ' hydroxyl group in the equatorial position. The synthesis of m-nitrobenzyl triphosphate, a new substrate of kanamycin nucleotidyltransferase, is reported. The kanamycin nucleotidyltransferase catalyzed reaction of kanamycin A with m-nitrobenzyl triphosphate is 2 orders of magnitude slower than that with ATP. The MALDI-TOF spectra of the purified products of both reactions revealed that kanamycin A was modified only at one position. The regiospecificity of the reaction catalyzed by kanamycin nucleotidyltransferase of kanamycin A with either ATP or m-nitrobenzyl triphosphate was determined directly by one- and two-dimensional hetero- and homonuclear NMR techniques. The site of the modification was unambiguously assigned to the 4' hydroxyl of kanamycin A; thus, the products formed are 4'-(adenosine-5'-phosphoryl)-kanamycin A and 4'-(m-nitrobenzyl phosphoryl)-kanamycin A. This eliminates the uncertainty concerning the point of modification since this could not be determined from the crystal structure of the enzyme with bound MgAMPCPP and kanamycin A [Pedersen, L. C., Benninig, M. M., and Holden, H. M. (1995) Biochemistry 34, 13305-13311].

Adenosine Monophosphate↗
PubMed2001

Cloning, overexpression, and purification of aminoglycoside antibiotic nucleotidyltransferase (2'')-Ia: conformational studies with bound substrates.

Aminoglycoside nucleotidyltransferase (2'')-Ia [ANT (2'')-Ia] was cloned from Pseudomonas aeruginosa and purified from overexpressing Escherichia coli BL21(DE3) cells. The first enzyme-bound conformation of an aminoglycoside antibiotic in the active site of an aminoglycoside nucleotidyltransferase was determined using the purified aminoglycoside nucleotidyltransferase (2' ')-Ia. The conformation of the aminoglycoside antibiotic isepamicin, a psuedo-trisaccharide, bound to aminoglycoside nucleotidyltransferase (2' ')-Ia has been determined using NMR spectroscopy. Molecular modeling, employing experimentally determined interproton distances, resulted in two different enzyme-bound conformations (conformer 1 and conformer 2) of isepamicin. Conformer 1 was by far the major conformer defined by the following average glycosidic dihedral angles: PhiBC = -65.26 +/- 1.63 degrees and PsiBC = -54.76 +/- 4.64 degrees. Conformer 1 was further subdivided into one major (conformer 1a) and two minor components (conformers 1b and 1c) based on the comparison of glycosidic dihedral angles PhiAB and PsiAB. The arrangement of substrates in the enzyme.metal-ATP.isepamicin complex was determined on the basis of the measured effect of the paramagnetic substrate analogue Cr(H2O)4ATP on the relaxation rates of substrate protons which were used to determine relative distances of isepamicin protons to the Cr3+. Both conformers of isepamicin yielded arrangements that satisfied the NOE restraints and the observed paramagnetic effects of Cr(H2O)4ATP. It has been suggested that aminoglycosides use both electrostatic interactions and hydrogen bonds in binding to RNA and that the contacts made by the A and B rings to RNA are the most important for binding [Fourmy, D., Recht, M. I., Blanchard, S. C., and Puglisi, J. D. (1996) Science 274, 1367-1371]. Comparisons based on the determined conformations of enzyme-bound aminoglycoside antibiotics also suggested that interactions of rings A and B with enzymes may be the major determinant in aminoglycoside binding to enzymes [Serpersu, E. H., Cox, J. R., DiGiammarino, E. L., Mohler, M. L., Ekman, D. R., Akal-Strader, A., and Owston, M. (2000) Cell Biochem. Biophys. (in press)]. The conformation of isepamicin bound to the aminoglycoside nucleotidyltransferase (2' ')-Ia, determined in this work, lent further support to this theory. Furthermore, comparison of enzyme-bound conformations of isepamicin to the RNA-bound conformation of gentamycin C1a also showed remarkable similarities between the enzyme-bound and RNA-bound aminoglycoside antibiotic conformations. These studies should aid in the design of effective inhibitors possessing a broad range of aminoglycoside-modifying enzymes as targets.

Bacterial Proteins↗
PubMed1974

Preparation of cells permeable to macromolecules by treatment with toluene: studies of transfer ribonucleic acid nucleotidyltransferase.

Transfer ribonucleic acid (tRNA) nucleotidyltransferase was studied after making cells permeable to macromolecules by treatment with toluene. The conditions of toluene treatment necessary for obtaining maximal activity were defined. Toluene treatment was most efficient when carried out for 5 min at 37 C at pH 9.0 on log-phase cells. No activity could be detected if cells were treated at 0 C, or in the presence of MgCl(2), or if the cells were in the stationary phase of growth. However, inclusion of lysozyme and ethylenediaminetetraacetic acid during the toluene treatment did render stationary phase cells permeable. The properties of tRNA nucleotidyltransferase from toluene-treated cells were essentially identical to those of purified enzyme with regard to pH optimum, specificity for nucleoside triphosphates and tRNA, and apparent K(m) values for substrates. In addition to tRNA nucleotidyltransferase, a variety of other enzymes which incorporate adenosine 5'-triphosphate into acid-precipitable material could also be detected in toluene-treated cells. Centrifugation of cells treated with toluene revealed that tRNA nucleotidyltransferase leaked out of cells, whereas other activities remained associated with the cell pellets. Chromatography of the material extracted from toluene-treated cells on Sephadex G-100 indicated that toluene treatment selectively extracts lower molecular weight proteins. The usefulness of such a procedure as an initial step in purification of such enzymes, and its application to tRNA nucleotidyltransferase, is discussed.

Adenosine↗
PubMed1987

tRNA nucleotidyltransferase is not essential for Escherichia coli viability.

The role of tRNA nucleotidyltransferase in Escherichia coli has been uncertain because all tRNA genes studied in this organism already encode the -C-C-A sequence. Examination of a cca mutant, originally thought to contain 1-2% enzyme activity, indicated that it actually produces an inactive fragment of 40 kd compared to 47 kd for the wild-type enzyme due to a nonsense mutation in its cca gene. To confirm that the residual activity in extracts of this strain is due to another enzyme, and that tRNA nucleotidyltransferase is non-essential, we have interrupted the cca gene in vitro, and transferred this mutant gene to a variety of strains. In all cases mutant strains are viable, although as much as 15% of the tRNA population contains defective 3' termini, and no tRNA nucleotidyltransferase is detectable. Mutant strains grow slowly, but can be restored to more normal growth by a relA mutation or by a decrease in RNase T activity. In the latter case the amount of defective tRNA decreases dramatically. These findings indicate that tRNA nucleotidyltransferase is not essential for E. coli viability, and therefore, that all essential tRNA genes in this organism encode the -C-C-A sequence.

Base Sequence↗
PubMed1966

Deoxyribonucleic acid nucleotidyltransferase from Landschütz ascites-tumour cells.

1. A purification procedure for DNA nucleotidyltransferase from Landschütz ascites-tumour cells is described. The enzyme can be separated from endogenous nucleic acid and from triphosphatase and deoxyribonuclease activities measurable at pH7.5. 2. The basic properties of the nucleotidyltransferase reaction are as follows. The enzyme has optimum activity at pH7.2-7.4. It displays an absolute requirement for DNA-primer, thermally-denatured DNA serving three to ten times as efficiently in this respect as native DNA. Maximum synthesis of polydeoxyribonucleotide occurs in the presence of all four deoxyribonucleoside 5'-triphosphates, but a limited incorporation of mononucleotide into polynucleotide is observed when the system is provided with only one triphosphate, or with various combinations of mono-, di- and tri-phosphates. The reaction requires the presence of a bivalent cation, and of those tested, Mg(2+) ions were by far the most effective. Manganous ions promoted synthesis but to a much smaller extent. Calcium ions did not support synthesis at all. At the appropriate concentrations, the univalent cations (sodium and potassium) stimulated the reaction by 25% and 125% respectively. The presence of EDTA in the reaction mixture stimulates the system five- to ten-fold. 3. The storage characteristics of the enzyme (as well as the activities of the various fractions) improve markedly if EDTA and 2-mercaptoethanol are included in the enzyme solution and in all preparative buffer solutions. 4. The enzyme loses more than 95% of its activity after heating for 1min. at 45 degrees . If the heating is conducted in the presence of DNA, the enzyme becomes relatively heat-resistant (presumably as a consequence of complex-formation with the DNA) and may actually display an activation effect. This is discussed in relation to a possible molecular conformation of the enzyme. 5. The product of the nucleotidyltransferase reaction is precipitable by acid or ethanol, and is susceptible to the actions of deoxyribonucleases I and II, snake-venom and spleen phosphodiesterases, and micrococcal nuclease. It forms a band in a density gradient of caesium chloride at a density similar to that of the DNA-primer. 6. By the criteria of nearest-neighbour frequency analyses, the product of the nucleotidyltransferase reaction has the characteristics to be expected of a polynucleotide synthesized in accordance with the template directions of the primer.

Chemical Phenomena↗
PubMed1982

Subcellular localization of the tRNA processing enzyme, tRNA nucleotidyltransferase, in Xenopus laevis oocytes and in somatic cells.

Earlier studies of the tRNA processing enzyme, tRNA nucleotidyltransferase, in rat liver cells suggested that the enzyme was absent from nuclei, whereas more recent studies with tRNA transcription-processing systems from Xenopus laevis oocytes indicated that the enzyme was present in germinal vesicles of these cells. In order to resolve this apparent discrepancy, the subcellular localization of tRNA nucleotidyltransferase in oocytes was directly determined, and its distribution in somatic cells was re-examined. Oocyte germinal vesicles, isolated manually, contained about 25% of the total tRNA nucleotidyltransferase activity of the cell, but the enzyme rapidly leaked out of these nuclei during storage. In contrast, purified nuclei isolated from rat or Xenopus liver using a variety of aqueous procedures were devoid of tRNA nucleotidyltransferase activity. Since rapidly-isolated, crude nuclear preparations from liver contained substantially more of the enzyme than could be accounted for by cytoplasmic contamination, it appears that the enzyme leaks out of the somatic cell nuclei during the lengthy times required for their purification. The implications of these results for the development of tRNA processing systems from somatic cell nuclei are discussed.

Animals↗
PubMed2001

A eubacterial origin for the human tRNA nucleotidyltransferase?

tRNA CCA-termini are generated and maintained by tRNA nucleotidyltransferases. Together with poly(A) polymerases and other enzymes they belong to the nucleotidyltransferase superfamily. However, sequence alignments within this family do not allow to distinguish between CCA-adding enzymes and poly(A) polymerases. Furthermore, due to the lack of sequence information about animal CCA-adding enzymes, identification of corresponding animal genes was not possible so far. Therefore, we looked for the human homolog using the baker's yeast tRNA nucleotidyltransferase as a query sequence in a BLAST search. This revealed that the human gene transcript CGI-47 (#AF151805) deposited in GenBank is likely to encode such an enzyme. To identify the nature of this protein, the cDNA of the transcript was cloned and the recombinant protein biochemically characterized, indicating that CGI-47 encodes a bona fide CCA-adding enzyme and not a poly(A) polymerase. This confirmed animal CCA-adding enzyme allowed us to identify putative homologs from other animals. Calculation of a neighbor-joining tree, using an alignment of several CCA-adding enzymes, revealed that the animal enzymes resemble more eubacterial ones than eukaryotic plant and fungal tRNA nucleotidyltransferases, suggesting that the animal nuclear cca genes might have been derived from the endosymbiotic progenitor of mitochondria and are therefore of eubacterial origin.

Amino Acid Sequence↗
PubMed1992

Cytoplasmic and mitochondrial tRNA nucleotidyltransferase activities are derived from the same gene in the yeast Saccharomyces cerevisiae.

ATP (CTP):tRNA-specific tRNA nucleotidyltransferase is an enzyme required for the synthesis of functional tRNAs in eukaryotic cells. Neither the tRNA genes in the nucleus nor in organelles encode the CCA end, so it must be added post-transcriptionally. The gene that codes for the enzyme that adds the CCA end to nuclear coded tRNAs in Saccharomyces cerevisiae has been isolated (Aebi, M., Kirchner, G., Chen, J.-Y., Vijayraghavan, U., Jacobson, A., Martin, N. C., and Abelson, J. (1990) J. Biol. Chem. 265, 16216-16220). We now demonstrate that there is a mitochondrial tRNA nucleotidyltransferase activity in yeast and that it is a matrix enzyme. A comparison of purified mitochondrial enzyme with its cytoplasmic counterpart revealed no differences. These results suggest that proteins responsible for this step in the maturation of tRNAs in the nucleus and mitochondria might be identical and coded by the same nuclear gene. Accumulation of shortened mitochondrial as well as cytoplasmic tRNAs in a strain with a temperature-sensitive tRNA nucleotidyltransferase is consistent with this hypothesis. Alteration of the wild type gene such that amino-terminal truncated proteins are produced leads to a defect in mitochondrial function and a decrease in mitochondrial nucleotidyltransferase activity. This provides a direct demonstration that one gene provides this enzyme activity for the biosynthesis of tRNAs in both the nuclear/cytoplasmic and mitochondrial compartments in yeast.

Amino Acid Sequence↗
PubMed1986

High-level overexpression, rapid purification, and properties of Escherichia coli tRNA nucleotidyltransferase.

The cloned Escherichia coli cca gene, described in the accompanying paper (Cudny, H., Lupski, J. R., Godson, G. N., and Deutscher, M. P. (1986) J. Biol. Chem. 261, 6444-6449), has been used to construct strains that overproduce tRNA nucleotidyltransferase, the enzyme that synthesizes the CCA terminus of tRNA. Strain UT481 (pEC4), which contains a 1.9-kilobase cca gene insert in plasmid pUC8, overproduces the enzyme by about 100-150-fold, probably under the control of the cca gene promoter. A second strain, containing a plasmid with a 1.5-kilobase insert, overproduces tRNA nucleotidyltransferase by about 650-fold, to a level of about 3-4% of the soluble cell protein. In this case, overexpression was dependent on the lac promoter of the plasmid. A rapid, two-step procedure was developed to purify large amounts of the enzyme from strain UT481 (pEC4) that was about 40% pure, free of ribonucleases, and suitable for use as a reagent for modification of tRNA molecules. Preparation of milligram quantities of homogeneous tRNA nucleotidyltransferase was accomplished by two further chromatographic steps. The structural and catalytic properties of this purified enzyme were similar to those from partially purified preparations previously described. The availability of large amounts of pure tRNA nucleotidyltransferase will not permit a variety of structural and functional studies of the enzyme that previously were not possible.

Cell Division↗
PubMed1976

Polyamine stimulation and cation requirements of rabbit liver tRNA nucleotidyltransferase.

We have examined the cation requirements of rabbit liver tRNA nucleotidyltransferase. The enzyme had an absolute requirement fo a divalent cation which could be satisfied by Mg2+, Mn2+ or Co2+. In contrast to the Escherichia coli enzyme, we have found no evidence to implicate Zn2+ in the action of rabbit liver tRNA nucleotidyltransferase. We have also identified a second cation requirement which was satisfied by divalent or monovalent cation or polyamines. The polyamines, spermine and spermidine, were most effective leading to 3-fold higher rates of nucleotide incorporation than could be obtained at any concentration of the other cations. However, the polyamines were not an absolute requirement for enzyme activity. The polyamine stimulation of tRNA nucleotidyltransferase was not due to alteration of the pH or ionic strength of the reaction mixture or reactivation of denatured tRNA or enzyme. Polyamines also had no effect on the apparent Km values of the substrates. Spermine increased the specificity of the enzyme for tRNA substrates and also inhibited the reverse action. Our results suggest that polyamines may be the normal counterions for tRNA in vivo, and that they affect the rate-limiting step in tRNA nucleotidyltransferase catalysis.

Animals↗
PubMed1986

Follicle-stimulating hormone and estrogen elevate deoxyribonucleic acid alpha-nucleotidyltransferase activity in relationship to deoxyribonucleic acid synthesis in immature rat ovaries.

The deoxyribonucleic acid polymerase activities and deoxyribonucleic acid contents of ovaries were measured to clarify the relationship between hormone-stimulated cell proliferation and the enzymes in ovaries of immature intact rats (4 to 29 days after birth) and hypophysectomized rats. The specific activity of deoxyribonucleic acid alpha-nucleotidyltransferase (the activity per microgram of deoxyribonucleic acid of the ovaries) drastically increased with an increase in the deoxyribonucleic acid content of the ovaries from 4 to 14 days after birth and then remained constant or slightly decreased after the increase, while the activity decreased gradually after hypophysectomy with no increase in the DNA content. Ovine follicle-stimulating hormone or estradiol-17 beta enhanced the deoxyribonucleic acid synthesis and alpha-nucleotidyltransferase activity in the ovaries of hypophysectomized rats, while deoxyribonucleic acid beta-nucleotidyltransferase activity showed no significant change. Ovine luteinizing hormone, progesterone, and testosterone caused no significant increases. These results suggest that follicle-stimulating hormone or estrogen causes the induction of deoxyribonucleic acid alpha-nucleotidyltransferase accompanied by deoxyribonucleic acid synthesis in cell proliferation in immature rat ovaries.

Animals↗
PubMed2005

Structure-guided mutational analysis of the nucleotidyltransferase domain of Escherichia coli NAD+-dependent DNA ligase (LigA).

NAD+-dependent DNA ligase (LigA) is essential for bacterial growth and a potential target for antimicrobial drug discovery. Here we queried the role of 14 conserved amino acids of Escherichia coli LigA by alanine scanning and thereby identified five new residues within the nucleotidyltransferase domain as being essential for LigA function in vitro and in vivo. Structure activity relationships were determined by conservative mutagenesis for the Glu-173, Arg-200, Arg-208, and Arg-277 side chains, as well as four other essential side chains that had been identified previously (Lys-115, Asp-117, Asp-285, and Lys-314). In addition, we identified Lys-290 as important for LigA activity. Reference to the structure of Enterococcus faecalis LigA allowed us to discriminate three classes of essential/important side chains that: (i) contact NAD+ directly (Lys-115, Glu-173, Lys-290, and Lys-314); (ii) comprise the interface between the NMN-binding domain (domain Ia) and the nucleotidyltransferase domain or comprise part of a nick-binding site on the surface of the nucleotidyltransferase domain (Arg-200 and Arg-208); or (iii) stabilize the active site fold of the nucleotidyltransferase domain (Arg-277). Analysis of mutational effects on the isolated ligase adenylylation and phosphodiester formation reactions revealed different functions for essential side chains at different steps of the DNA ligase pathway, consistent with the proposal that the active site is serially remodeled as the reaction proceeds.

Alanine↗
PubMed1977

Preparation of Escherichia coli tRNAs terminating of modified nucleosides by the use of CTP(ATP):tRNA nucleotidyltransferase and polynucleotide phosphorylase.

Two procedures were investigated for the modification of tRNAs at the 3'-terminal nucleoside. The first involved the incubation of an enzymatically abreviated tRNA (tRNA-C-COH) with appropriate nucleoside triphosphates in the presence of CTP(ATP):tRNA nucleotidyltransferase from Escherichia coli and yeast. The E. coli enzyme did not utilize 2'- or 3'-deoxyadenosine 5'-triphosphate as substrates, but affected incorporation of the 2'- and 3'-O-methyladenosine triphosphates onto tRNA-C-Cou to the extent of 30 and 37%, respectively. Although incorporation of the deoxynucleotides could not be effected using the E. coli enzyme, yeast CTP(ATP:tRNA nucleotidyltransferase produced the desired tRNAs in yields of 45-65%. The second modification procedure involved incubation of tRNA-C-COH with (appropriately blocked) nucleoside diphosphates in the presence of polynucleotide phosphorylase. This procedure afforded the tRNAs terminating in 2'- and 3'-deoxyadenosine in yields of 4% (and the yield of the former was increased to 36% when the incubation was carried out in the presence of 20% methanol). The yields of tRNAs terminating in 2'- and 3'-O-methyladenosing produced by this procedure were 55 and 17%, respectively. Because only single isomers of most of the tRNAs terminating in 2'- and 3'-deoxy- and O-methyladenosine are aminoacylated, attempts were made to obtain the other isomericaminoacyl-tRNA by enzymatic introduction of chemically preaminoacylated nucleotides onto tRNA-C-COH. Although incubation of tRNA-C-COH with three aminoacylated nucleoside 5'-triphosphates and E. coli CTP(ATP):tRNA nucleotidyltransferase did not result in production of the desired tRNAs to a detectable extent, incubation with 2'-deoxy-3'-O-L-phenylalanyladenosine 5'-diphosphate and polynucleotide phosphorylase afforded E. coli tRNA terminating with the corresponding aminoacylated deoxynucleoside.

Adenosine Diphosphate↗