Photoanalogues of the Initiation Substrates of the RNA Polymerase II, 5‐Azido‐2‐Nitrobenzoyl Derivatives of the ATP γ‐Amidophosphate: The Possible Photoinduced Degradation of the Functional Group to an N‐Arylhydroxylamine

Photoanlogues of the initiation substrates of the RNA polymerase II, N₃Ar‐ NH(CH₂)_nNHpppA where N₃Ar is 5‐azido‐2‐nitrobenzoyl group (n = 2 or 4) were synthesized, allowing the preparation of photoreactive oligonucleotides in situ by RNA polymerase II for application as photolabels. Photolysis of p‐nitro‐substituted aromatic azide in aqueous medium was investigated. Using the azoxy‐coupling reaction it was possible to determine whether a nitrene or p‐nitrophenyl hydroxylamine azoxy compound is the trappable intermediate that is generated at ambient temperature in aqueous solution.     

Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly.

Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II.     

Phylogenetic analysis of norovirus isolates involved in some Canadian gastroenteritis outbreaks in 2004 and 2005

Noroviruses are recognized as the most common cause of nonbacterial gastroenteritis worldwide. In this study, we investigated the molecular epidemiology of noroviral isolates in Canada from 2004 to 2005 by sequencing the RNA polymerase gene and capsid N-terminal/shell (N/S) domain. Norovirus genogroups I and II were thus found to have co-circulated in Canada during the studied period, with a higher incidence of genogroup II (95.7%). The GII-4 or Lordsdale subgroup was the predominant genotype, suggesting that norovirus genogroup II is the major cause of viral gastroenteritis in Canada, as it is in many other countries. Phylogenetic analyses of the RNA polymerase gene and the capsid N/S domain indicated different genotypes for 2 strains, suggesting probable genetic recombination. Sequencing of the norovirus polymerase gene may reflect actual classification but should be supported by sequence information obtained from the capsid gene.     

RNA polymerase II acts as an RNA‐dependent RNA polymerase to extend and destabilize a non‐coding RNA

RNA polymerase II (Pol II) is a well‐characterized DNA‐dependent RNA polymerase, which has also been reported to have RNA‐dependent RNA polymerase (RdRP) activity. Natural cellular RNA substrates of mammalian Pol II, however, have not been identified and the cellular function of the Pol II RdRP activity is unknown. We found that Pol II can use a non‐coding RNA, B2 RNA, as both a substrate and a template for its RdRP activity. Pol II extends B2 RNA by 18 nt on its 3′‐end in an internally templated reaction. The RNA product resulting from extension of B2 RNA by the Pol II RdRP can be removed from Pol II by a factor present in nuclear extracts. Treatment of cells with α‐amanitin or actinomycin D revealed that extension of B2 RNA by Pol II destabilizes the RNA. Our studies provide compelling evidence that mammalian Pol II acts as an RdRP to control the stability of a cellular RNA by extending its 3′‐end.     

Comparison of the effects of polyamines on the activities of RNA polymerases from murine normal tissues and transplantable tumors

Nuclear DNA-dependent RNA polymerases I, II and III were purified from kidney, liver and spleen from Swiss mice (Mus musculis) and from seven transplantable murine tumors. In the presence of the optimal concentration of (NH4)2SO4 for each polymerase, 1-8 mM spermidine or spermine stimulated most polymerases several fold, and generally, enzyme I was stimulated more than either enzyme II or III. Spermine was more efficacious than spermidine as a stimulant of polymerase activity except for polymerase III from three tumors. Tumor polymerases I (or II) and the corresponding normal tissue enzymes responded similarly to the polyamines. Stimulation of a RNA polymerase by a polyamine could not be correlated with the growth rate of the tissues of polymerase origin or with the tissue's RNA polymerase or RNA synthetic activities.     

The sensitivity of RNA polymerases I and II from Novikoff hepatoma (N1S1) cells to 3''-deoxyadenosine 5''-triphosphate.

The synthesis of ribosomal precursor RNA in Novikoff hepatoma (N1S1) cells is very sensitive to cordycepin (3'-dA). The synthesis of hnRNA, however, is resistant to inhibition concentrations of 3'-dA that completely block the synthesis of 45S ribosomal RNA precursor. We have examined the RNA polymerases present in these cultured cells with regard to their sensitivity to cordycepin 5'-triphosphate (3'-dATP) in an effort to explain the differential inhibition of RNA synthesis observed in vivo. RNA polymerases I and II were characterized on the basis of their chromatographic behavior on DEAE-Sephadex, as well as the response of their enzymatic activities to ionic strength, the divalent metal ions Mn2+ and Mg2+, and the toxin alpha-amanitin. For both enzymes the inhibition of in vitro RNA synthesis by 3'-dATP was competitive for ATP. The km values for ATP and the K1 values for 3'-dATP for the two enzymes were quite similar. RNA polymerase II, the enzyme presumed responsible for hnRNA synthesis, was actually slightly more sensitive to 3'-dATP than RNA polymerase I, the enzyme presumed responsible for ribosomal precursor RNA synthesis. Similar data were obtained when the RNA polymerases were assayed in isolated nuclei. These results indicate that the differential inhibition of RNA synthesis caused by 3'-dA in vivo cannot be simply explained by differential sensitivity of RNA polymerases I and II to 3'-dATP.     

Selective Modulation of RNA Polymerase I Activity during Growth Transitions in the Soybean Seedling

RNA polymerase I and II activities were measured in tissues of the soybean (Glycina max, var. Wayne) hypocotyl where dramatic changes in the relative level of RNA synthesis are associated with normal and auxin-induced growth transitions. When assayed in isolated nuclei, the activity of RNA polymerase I changed much more than the activity of RNA polymerase II during these growth transitions. The activity of RNA polymerase I expressed in the nuclei generally showed a positive correlation with the relative level of RNA synthesis (i.e. accumulation) of that tissue. Following solubilization of the RNA polymerases from these isolated nuclei and fractionation of them on DEAE-cellulose, the activity of RNA polymerase I relative to that of RNA polymerase II showed smaller changes during these growth transitions than when assayed in the nuclei. Thus, these data indicate that the activity of RNA polymerase I is significantly modulated in the nucleus, up or down depending upon the growth state, during growth transitions in the soybean in addition to lesser changes which occur in the apparent level of the enzyme.     

Fluorescence resonance energy transfer studies on the proximity relationship between the intrinsic metal ion and substrate binding sites of Escherichia coli RNA polymerase

DNA-dependent RNA polymerase from Escherichia coli contains 2 mol of zinc/mol of holoenzyme (alpha 2 beta beta' sigma) with one zinc each in the beta and beta' subunits. A new method to substitute selectively the zinc in the beta subunit was developed by the inactivation of RNA polymerase with 0.25 M NaNO3, 1 M NaCl, 1 mM diaminocyclohexane tetraacetic acid, and 0.1 mM dithiothreitol followed by reconstitution with Co(II), Cd(II), or Cu(II). The hybrid Co-Zn, Cd-Zn, or Cu-Zn RNA polymerase thus obtained retains, respectively, 91, 88, and 50% enzyme activity of the reconstituted Zn-Zn RNA polymerase. Co-Zn RNA polymerase exhibits absorption maxima at 395 and 465 nm, and Cu-Zn RNA polymerase at 637 nm (epsilon = 815 M-1 cm-1). 1-Aminonaphthalene-5-sulfonic acid (AmNS) derivatives of ATP, UTP, and dinucleoside monophosphates (diNMPs), UpA or ApU, were synthesized with AmNS attached to NTP via a gamma-phosphoamidate bond or to diNMPs via a 5'-secondary amine linkage. Since the fluorescence emission maxima of (5'-AmNS)UpA, (gamma-AmNS)ATP, and (gamma-AmNS)UTP at 445, 464, and 464 nm, respectively, when excited at 340 nm, overlap the 465-nm absorption band of Co-Zn RNA polymerase, the spatial relationship between fluorescence substrate analogs and the intrinsic Co(II) in Co-Zn RNA polymerase was studied by fluorescence resonance energy transfer technique. The fluorescence of the initiator, (5'-AmNS)UpA, and elongator, (gamma-AmNS)UTP, of the RNA chain, was quenched 20.3 and 7.1%, by the addition of saturation concentration of Zn-Zn RNA polymerase, and 21.3 and 14.7%, respectively, by the addition of template, poly(dA-dT). The fluorescence of (5'-AmNS)UpA and (gamma-AmNS)UTP was quenched 81.8 and 80.6%, respectively, by the addition of the saturation concentration of Co-Zn RNA polymerase in the absence of template, and 82.7 and 82.9% in the presence of template. On the basis of respective Ro values of 21.3 and 21.9 A for the (5'-AmNS)UpA-Co and (gamma-AmNS)UTP-Co pairs, the distances from Co(II) to the initiation site and to the elongation site were calculated to be 17.4 and 17.5 A, respectively, in the absence and 17.2 and 17.4 A in the presence of template.     

Phosphorylation of rat C6 glioma cell DNA-dependent RNA polymerase II in vivo. Identification of phosphorylated subunits and modulation of phosphorylation by isoproterenol and N6,O2'-dibutyryl cyclic AMP

Evidence is presented that isoproterenol treatment of rat C6 glioma cells, under conditions that increase glioma cell cAMP levels, causes the phosphorylative modification of several RNA polymerase II subunits. RNA polymerase II in control and isoproterenol-stimulated 32Pi-labeled confluent glioma cells was immunoprecipitated from ribonuclease-treated nuclear extracts with hen anti-calf RNA polymerase II antiserum conjugated to Sepharose. The immunoprecipitated RNA polymerase II was analyzed for 32P-labeled subunits by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Using this technique, we have shown that isoproterenol causes a time-dependent increase of phosphate incorporation into RNA polymerase II subunits of 214,000, 180,000, 140,000, 35,000, 28,000, and 16,500 daltons. Phosphate incorporation occurred exclusively on serine in all of the six subunits. About 0.5-2 mol of phosphate/mol of RNA polymerase II subunit were incorporated. Dibutyryl cAMP (10(-3)M) mimics the stimulatory action of isoproterenol and mediates increased phosphate incorporation into the six subunits. (RS)-propranolol (10(-4)M) prevents the isoproterenol-mediated phosphorylative changes. These data indicate that isoproterenol, via cAMP, mediates a transient structural modification of RNA polymerase II subunits in rat C6 glioma cells which may possibly lead to a modulation of RNA polymerase II function(s).