Two distinct poly(A) polymerases isolated from the cytoplasm of Ehrlich ascites tumour cells

The poly(A) polymerases from the cytosol and ribosomal fractions of Ehrlich ascites tumour cells are isolated and partially purified by DEAE-cellulose and phosphocellulose column chromatography. Two distinct enzymes are identified: (a) a cytosol Mn2+-dependent poly(A) polymerase (ATP:RNA adenylyltransferase) and (b) a ribosome-associated enzyme defined tentatively as ATP(UTP): RNA nucleotidyltransferase. The cytosol poly(A) polymerase is strictly Mn2+-dependent (optimum at 1 mM Mn2+) and uses only ATP as substrate, poly(A) is a better primer than ribosomal RNA. The purified enzyme is free of poly(A) hydrolase activity, but degradation of [3H]poly(A) takes place in the presence of inorganic pyrophosphate. Most likely this enzyme is of nuclear origin. The ribosomal enzyme is associated with the ribosomes but it is found also in free state in the cytosol. The purified enzyme uses both ATP and UTP as substrates. The substrate specificity varies depending on ionic conditions: the optimal enzyme activity with ATP as substrate is at 1 mM Mn2+, while that with UTP as substrate is at 10--20 mM Mg2+. The enzymes uses both ribosomal RNA and poly(A) [but not poly(U)] as primers. The purified enzyme is free of poly(A) hydrolase activity.     

A critical switch in the enzymatic properties of the Cid1 protein deciphered from its product-bound crystal structure

The addition of uridine nucleotide by the poly(U) polymerase (PUP) enzymes has a demonstrated impact on various classes of RNAs such as microRNAs (miRNAs), histone-encoding RNAs and messenger RNAs. Cid1 protein is a member of the PUP family. We solved the crystal structure of Cid1 in complex with non-hydrolyzable UMPNPP and a short dinucleotide compound ApU. These structures revealed new residues involved in substrate/product stabilization. In particular, one of the three catalytic aspartate residues explains the RNA dependence of its PUP activity. Moreover, other residues such as residue N165 or the β-trapdoor are shown to be critical for Cid1 activity. We finally suggest that the length and sequence of Cid1 substrate RNA influence the balance between Cid1''s processive and distributive activities. We propose that particular processes regulated by PUPs require the enzymes to switch between the two types of activity as shown for the miRNA biogenesis where PUPs can either promote DICER cleavage via short U-tail or trigger miRNA degradation by adding longer poly(U) tail. The enzymatic properties of these enzymes may be critical for determining their particular function in vivo.     

Cid13 is a cytoplasmic poly(A) polymerase that regulates ribonucleotide reductase mRNA

Fission yeast Cid13 and budding yeast Trf4/5 are members of a newly identified nucleotidyltransferase family conserved from yeast to man. Trf4/5 are thought to be essential DNA polymerases. We report that Cid13 is a poly(A) polymerase. Unlike conventional poly(A) polymerases, which act in the nucleus and indiscriminately polyadenylate all mRNA, Cid13 is a cytoplasmic enzyme that specifically targets suc22 mRNA that encodes a subunit of ribonucleotide reductase (RNR). cid13 mutants have reduced dNTP pools and are sensitive to hydroxyurea, an RNR inhibitor. We propose that Cid13 defines a cytoplasmic form of poly(A) polymerase important for DNA replication and genome maintenance.     

Mutation analysis of the GDD sequence motif of a calicivirus RNA-dependent RNA polymerase

The RNA-dependent RNA polymerase from rabbit hemorrhagic disease virus, a calicivirus, is known to have a conserved GDD amino acid motif and several additional regions of sequence homology with all types of polymerases. To test whether both aspartic acid residues are in fact involved in the catalytic activity and metal ion coordination of the enzyme, several defined mutations have been made in order to replace them by glutamate, asparagine, or glycine. All six mutant enzymes were produced in Escherichia coli, and their in vitro poly(U) polymerase activity was characterized. The results demonstrated that the first aspartate residue was absolutely required for enzyme function and that some flexibility existed with respect to the second, which could be replaced by glutamate.     

Poly(A) Polymerase from Vaccinia Virus-Infected Cells I. Partial Purification and Characterization

Poly(A) polymerase activity is induced during vaccinia virus infection of HeLa cells. The enzyme is maximally induced at 3.5 h postinfection. Partial purification frees the preparation of RNase activity and RNA polymerase activity. ATP is the substrate for poly(A) synthesis. A small amount of poly(A) is produced from added adenosine diphosphate due to the production of ATP by an adenylate kinase present in the preparation. The incorporation of ATP into poly(A) is dependent on divalent cations (Mg²⁺ or Mn²⁺) and is not inhibited by UTP, CTP, or GTP. Poly(U) stimulates ATP incorporation; poly(A) and poly(C) have little effect on ATP incorporation, and poly(dT) is extremely inhibitory. RNA prepared from HeLa cells and from the partially purified poly(A) polymerase (the enzyme preparation contains endogenous RNA [Brakel and Kates]) stimulates ATP incorporation by poly(A) polymerase which was subjected to DEAE-cellulose chromatography. RNase's, pancreatic and T₁, inhibit the production of poly(A). DNase has little effect. Poly(U) is able to stimulate poly(A) production in the presence of T₁ RNase.     

The Cid1 poly(U) polymerase

The Schizosaccharomyces pombe cytoplasmic protein Cid1 acts as a poly(U) polymerase (PUP). Polyadenylated actin mRNA, a target of this activity, is uridylated upon arrest in S phase and is likely to be one of many such Cid1 targets. This RNA uridylation pathway appears to be conserved, as Cid1 orthologs in Arabidopsis thaliana, Caenorhabditis elegans and humans display PUP activity either in vitro or in Xenopus laevis oocytes. Here, we review the literature on Cid1, other PUPs and uridylation, a conserved and previously under-appreciated mechanism of RNA regulation.     

Mutation of the aspartic acid residues of the GDD sequence motif of poliovirus RNA-dependent RNA polymerase results in enzymes with altered metal ion requirements for activity.

The poliovirus RNA-dependent RNA polymerase, 3Dpol, is known to share a region of sequence homology with all RNA polymerases centered at the GDD amino acid motif. The two aspartic acids have been postulated to be involved in the catalytic activity and metal ion coordination of the enzyme. To test this hypothesis, we have utilized oligonucleotide site-directed mutagenesis to generate defined mutations in the aspartic acids of the GDD motif of the 3Dpol gene. The codon for the first aspartate (3D-D-328 [D refers to the single amino acid change, and the number refers to its position in the polymerase]) was changed to that for glutamic acid, histidine, asparagine, or glutamine; the codons for both aspartic acids were simultaneously changed to those for glutamic acids; and the codon for the second aspartic acid (3D-D-329) was changed to that for glutamic acid or asparagine. The mutant enzymes were expressed in Escherichia coli, and the in vitro poly(U) polymerase activity was characterized. All of the mutant 3Dpol enzymes were enzymatically inactive in vitro when tested over a range of Mg2+ concentrations. However, when Mn2+ was substituted for Mg2+ in the in vitro assays, the mutant that substituted the second aspartic acid for asparagine (3D-N-329) was active. To further substantiate this finding, a series of different transition metal ions were substituted for Mg2+ in the poly(U) polymerase assay. The wild-type enzyme was active with all metals except Ca2+, while the 3D-N-329 mutant was active only when FeC6H7O5 was used in the reaction. To determine the effects of the mutations on poliovirus replication, the mutant 3Dpol genes were subcloned into an infectious cDNA of poliovirus. The cDNAs containing the mutant 3Dpol genes did not produce infectious virus when transfected into tissue culture cells under standard conditions. Because of the activity of the 3D-N-329 mutant in the presence of Fe2+ and Mn2+, transfections were also performed in the presence of the different metal ions. Surprisingly, the transfection of the cDNA containing the 3D-N-329 mutation resulted in the production of virus at a low frequency in the presence of FeSO4 or CoCl2. The virus derived from transfection in the presence of FeSO4 grew slowly, while the viruses recovered from transfection in CoCl2 grew at a rate which was similar to that of the wild-type poliovirus.(ABSTRACT TRUNCATED AT 400 WORDS)     

A template independent, rifampicin sensitive poly (A).poly (U) synthesizing activity present in Bacillus subtilis.

A template independent poly (A)·poly (U) synthesizing activity has been isolated from $\text{Bacillus subtilis}$. This activity is eluted from a DNA-cellulose column along with DNA-dependent RNA polymerase. The column fractions which exhibit this activity contain RNA polymerase holoenzyme plus a polypeptide which is slightly larger than sigma factor; pure RNA polymerase holoenzyme did not synthesize poly (A)·poly (U). The activity was dependent on the presence of ATP, UTP, and Mn⁺⁺ (Mg⁺⁺ could not substitute), and was inhibited by rifampicin, streptolydigin, and Cibacron Blue. The incorporation of nucleotides was not linear with time, but appeared after a lag period. The results suggest that a modified form of DNA-dependent RNA polymerase analogous to $\text{Escherichia coli}$ holoenzyme II is catalyzing the synthesis of poly (A)·poly (U).     

Probing histidine-substrate interactions in tyrosyl-tRNA synthetase using asparagine and glutamine replacements

We have analyzed the interactions of a histidine residue with a substrate using site-directed mutagenesis. Previous studies on tyrosyl-tRNA synthetase from Bacillus stearothermophilus have shown that a histidine residue (His-48) makes an interaction with ATP, which is improved on mutating Thr-51----Pro-51. We find on replacing His-48 in wild-type enzyme with either asparagine or glutamine that Asn-48 is equally as good as His-48 but His-48----Gln-48 leads to a far lower activity. The side chain of an asparagine residue may be superimposed on that of a histidine so that the amide-NH2 group of asparagine occupies the same position as the pi-N of histidine, whereas the equivalent -NH2 group of glutamine may be superimposed upon the tau-N. This suggests that it is the pi-N of histidine that hydrogen bonds with ATP and that there is no significant electrostatic interaction between the histidine and ATP. Incorporating the Pro-51 mutation into each of the Asn-48 and Gln-48 mutants gives an improvement in the affinity of the enzyme for ATP, but this improvement is less than that seen with the wild-type enzyme.     

Inhibition of a novel subspecies of DNA polymerase α by 2′-deoxy-2′-azidoadenosine 5′-triphosphate

DNA polymerase α₁, a subspecies of DNA polymerase α of Ehrlich ascites tumor cells, was associated with a novel RNA polymerase activity and utilized poly(dT) and single-stranded circular fd DNA as a template without added primer in the presence of ribonucleoside triphosphates and a specific stimulating factor. DNA synthesis in the above system was inhibited by the ATP analogue, 2′-deoxy-2′-azidoadenosine 5′-triphosphate more than the DNA synthesis with poly(dT)·oligo(rA) by DNA polymerase α₁ and RNA synthesis by mouse RNA polymerases I and II. Kinetic analysis showed that the analogue inhibited DNA polymerase α₁ activity on poly(dT) competitively with respect to ATP, suggesting that the analogue inhibited RNA synthesis by the associated RNA polymerase activity.     

2'-Deoxy-2'-azidoadenosine triphosphate and 2'-deoxy-2'-fluoroadenosine triphosphate as substrates and inhibitors for Escherichia coli DNA-dependent RNA polymerase

The effects of 2'-substitutions of ATP on the substrate and inhibitor properties for RNA synthesis were studied in the poly(dAT)-dependent reaction of Escherichia coli RNA polymerase. In the presence of UTP, 2'-deoxy-2'-azidoadenosine 5'-triphosphate (AZTP) was incorporated into an acid-insoluble fraction at one-tenth of the rate of ATP incorporation; it thus acts as a competitive inhibitor for poly(AU) synthesis. On the other hand, another ATP analog, 2'-deoxy-2'-fluoroadenosine 5'-triphosphate (AfTP), was co-polymerized with UTP into acid-insoluble materials at a rate less than 1% of that of ATP incorporation; in addition, it exerted a strong but mixed-type inhibition on poly(AU) synthesis. Different modes of action of the two ATP analogs are discussed in connection with the specificity of substrate recognition by RNA polymerase.     

Properties of unprimed poly(A)-poly(U) synthesis by Caulobacter crescentus RNA polymerase.

Some properties of unprimed poly(A)-poly(U) synthesis by DNA-dependent RNA polymerase from Caulobacter crescentus were examined. The reaction required ATP and UTP as substrates and manganese as a divalent cation. Rifampicin completely inhibited the reaction at a concentration of 1 micron/ml, and the enzyme catalyzed the polymer synthesis well regardless of the presence of GTP, CTP or both. The chain length of the poly(A)-poly(U) synthesized was about one hundred base pairs, as estimated from a sedimentation velocity and the molar ratio of [3H]AMP to [gamma-32P]ATP incorporated into the poly(A)-poly(U). The reaction was dependent on the square of the enzyme concentration and the enzyme dimers formed complexes with poly(A)-poly(U) during the reaction.