High Concentrations Of Α-amanitin Research Articles

A Chloroplastic RNA Polymerase Resistant to Tagetitoxin Is Involved in Replication of Avocado Sunblotch Viroid

Avocado sunblotch viroid (ASBVd), the type species of the family Avsunviroidae, replicates and accumulates in the chloroplast. Two main chloroplastic RNA polymerases have been described: the plastid-encoded polymerase (PEP) with a multisubunit structure similar to the Escherichia coli enzyme and a single-unit nuclear-encoded polymerase (NEP) resembling phage RNA polymerases. On a different basis, sensitivity to tagetitoxin, two major RNA polymerase activities, tagetitoxin sensitive (TS) and resistant (TR), have been found in plastids. The most plausible candidates for the TS and TR RNA polymerases are PEP and NEP, respectively. To gain an insight into the enzymology of the polymerization of ASBVd strands, purified chloroplast preparations from ASBVd-infected leaves were assayed for their in vitro ability to transcribe ASBVd RNAs together with some representative genes (psbA, 16SrDNA, accD, and rpoB) of the three classes of chloroplastic genes according to their promoter structure. High concentrations of α-amanitin had no effect on gene or on viroid transcription, but tagetitoxin (5–10 μM) prevented transcription of all these genes without affecting synthesis of ASBVd strands; only at higher tagetitoxin concentrations (50–100 μM) was a 25% inhibition observed. These results suggest that NEP is the RNA polymerase required in ASBVd replication, although the participation of another TR RNA polymerase from the chloroplast cannot be excluded.

Synthesis of dinucleoside tetraphosphates by RNA polymerase B (II) from calf thymus

Highly purified RNA polymerase B (II) from calf thymus catalyses the synthesis of dinucleoside tetraphosphates from ribonucleoside triphosphates in the absence of an oligonucleotide primer or additional protein factors. The reaction requires a DNA template and bivalent cations such as Mn 2+ or Mg 2+. It is strongly inhibited by heparin and high concentrations of α-amanitin but not by rifampicin. On a given template various dinucleoside tetraphosphates of different sequence are formed although the yield depends on the nature of the template.

Structural relationships among the multiple forms of DNA-dependent RNA polymerase II from cultured parsley cells.

DNA-dependent RNA polymerase II (or B) was purified from cultured parsley cells, and its molecular structure was examined in detail. Upon centrifugation through glycerol gradients, RNA polymerase II sediments as a single band with an apparent sedimentation constant of 15 S. No contamination with RNA polymerases I or III could be detected when the activity of purified RNA polymerase II was assayed in the presence of high concentrations of α-amanitin. Analysis of purified RNA polymerase II by nondenaturing and denaturing polyacrylamide gel electrophoresis revealed that this enzyme exists in multiple forms. They were designated IIo, IIA, and IIB. It is suggested that each form has a subunit of Mr= 140 000 as well as smaller polypeptides in common. They differ, however, in the molecular weights of their largest subunits which is 220 000 in form IIo, 200 000 in form IIA, and 180 000 in form IIB. These large subunits were labelled with 125I, digested with trypsin, and tryptic digests were compared by two-dimensional analysis on thin-layer plates [Elder et al. (1977) J. Biol. Chem. 252, 6510–6515]. Fingerprints of tryptic digests from the polypeptides with Mr= 220 000, Mr= 200 000, and Mr= 180 000 were similar. It is, therefore, suggested that these subunits are structurally related. A tryptic digest was also produced from the subunit with Mr= 140 000. Its fingerprint was found to yield a considerably different distribution of peptides as compared to those from the three large subunits.

Primase initiates Okazaki pieces during polyoma DNA synthesis

SHORT fragments (Okazaki pieces) are formed as intermediates during elongation of nascent DNA strands1. In isolated nuclei from polyoma-virus infected 3T6 cells Okazaki pieces are initiated by a short RNA primer (initiator RNA, iRNA) with the approximate size of a decanucleotide2. The nature of the polymerase responsible for primer synthesis is not known, but high concentrations of α-amanitin, a drug which inhibits both RNA polymerase II (ref. 3) and III (ref. 4), have no effect on primer formation (unpublished results). We now present evidence that iRNA is not synthesised by any of the known RNA polymerases, but that, instead, a mammalian enzyme is involved which in its action corresponds to the dnaG gene product of E. coli. The bacterial enzyme was named primase5. It participates in the initiation of DNA strands of some single-stranded bacteriophages and has been suggested to be involved in the synthesis of bacterial Okazaki pieces6,7. One of the distinguishing features of the E. coli enzyme is its ability to use both ribonucleotides and deoxyribonucleotides for primer synthesis5,8.

A new class of small nuclear RNA molecules synthesized by a type I RNA polymerase in hela cells

A new class of previously undetected small RNA molecules with a range of discrete sizes between 6S and 10S has been identified in HeLa cell nuclei. They differ in size and location from the previously described small nuclear RNA species (snRNA). These RNA molecules were initially found by selective RNA labeling in vitro in isolated nuclei. The in vitro products migrate in gel electrophoresis in the region from 6–10S with predominant components between 8S and 10S. They are labeled in the presence of very high concentrations of α-amanitin (150–400 μg/ml), suggesting they are synthesized by a type I polymerase. Unlike the major polymerase I product, ribosomal precursor RNA, however, these molecules are found in the nucleoplasm and their labeling is not affected by pretreatment of cells with low concentrations of actinomycin D (0.04 μg/ml). Their formation by a presumptive polymerase I type of enzyme is the basis of their tentative designation as small nuclear polymerase I (snPI) RNAs. The snPI RNA molecules appear to be associated with chromatin and the nuclear matrix. They can be selectively eluted from nuclei leaving most of hnRNA behind. This association is used as the basis of fractionation procedures which separate these molecules from hnRNA and permit the demonstration of the synthesis of at least the most predominant of these RNA molecules in vivo. w

DNA-Dependent RNA Polymerase C. Occurrence and Localization in Various Animal Cells

DNA-dependent RNA-polymerase C, initially shown to exist in rat liver tissue and characterized by its sensitivity toward high concentrations of α-amanitin, has been shown to exist in HeLa cells and to be present in variable amounts in the nucleus of the rat liver cell. Enzyme C has been shown to co-chromatograph with nucleolar RNA polymerase A (or I) on DEAE-cellulose and can clearly be separated from the latter by chromatography on DEAE-Sephadex. It is possible that RNA polymerase C may occur in multiple forms although the significance of this finding is unclear at present. From its similar chromatographic properties and sensitivity toward α-amanitin it is possible that RNA polymerase C corresponds to polymerase III.

Purification and properties of DNA-dependent RNA polymerases from yeast.

A procedure for the large‐scale purification of yeast RNA polymerases A and B is described. The final enzyme preparations obtained are fairly homogeneous, as revealed by electrophoresis in polyacrylamide gels containing sodium dodecylsulfate. Both enzymes contain two large subunits with molecular weights of about 190000 and 135000 for polymerase A and 175000 and 140000 for polymerase B in a 1:1 ratio. The third polymerase from yeast (enzyme C) has not yet been sufficiently purified to determine its subunit structure. All three RNA polymerases require four ribonucleoside triphosphates, DNA and Mn2+ for activity. Denatured DNA is preferred over native DNA as template. Ammonium sulfate in low concentrations (20 to 50 mM) stimulates the enzyme activities two to three‐fold; high concentrations are inhibitory. Only RNA polymerase 3 is sensitive towards α‐amanitin. This enzyme is almost totally inhibited by a concentration of 100 μg/ml of the drug, whereas polymerases A and C remain virtually unaffected even at high concentrations of α‐amanitin. The RNA polymerases from yeast have, in general, many properties in common with the corresponding enzymes of multicellular organisms.