amanitins and tagetitoxin

Genome projects involving Leishmania and other trypanosomatids have revealed that most genes in these organisms are organized into large clusters of genes on the same DNA strand. We have previously shown that transcription of the entire Leishmania major Friedlin (LmjF) chromosome 1 (chr1) initiates bidirectionally between two divergent gene clusters. Here, we analyze transcription of LmjF chr3, which contains two convergent clusters of 67 and 30 genes, separated by a tRNA gene, with a single divergent protein-coding gene located close to the "left" telomere. Nuclear run-on analyses indicate that specific transcription of chr3 initiates bidirectionally between the single subtelomeric gene and the adjacent 67-gene cluster, close to the "right" telomere upstream of the 30-gene cluster, and upstream of the tRNA gene. Transcription on both strands terminates within the tRNA-gene region. Transient-transfection studies support the role of the tRNA-gene region as a transcription terminator for RNA polymerase II (Pol II) and Pol III, and also for Pol I.

Topics: Amanitins; Animals; Artificial Gene Fusion; Base Sequence; Chromosomes; Dicarboxylic Acids; DNA-Directed RNA Polymerases; DNA, Intergenic; DNA, Single-Stranded; Genes, Protozoan; Leishmania; Luciferases; Molecular Sequence Data; Multigene Family; Nucleic Acid Hybridization; Organophosphorus Compounds; RNA, Messenger; RNA, Transfer, Lys; Transcription, Genetic; Ultraviolet Rays

The transcriptional activity of nucleoids changes during plastid development, presumably due to the morphological and molecular differences of the nucleoids. Pea chloroplast nucleoids have an abundant 70-kDa protein identified as sulfite reductase (SiR) that can compact DNA. Using an in vitro transcription assay, we show here that heparin increased the transcriptional activity of chloroplast nucleoids with concomitant release of SiR. Using a fluorometric method we developed for analyzing DNA compaction, we found that the fluorescence intensity of chloroplast DNA stained with 4',6-diamidino-2-phenylindole was decreased by the addition of SiR and increased by the subsequent addition of heparin. Addition of exogenous SiR increased the compaction of isolated nucleoids, and the addition of heparin relaxed it. SiR effectively repressed the in vitro transcription activity of nucleoids and counteracted the activation by heparin. These results suggest that SiR regulates the transcriptional activity of chloroplast nucleoids through changes in DNA compaction.

Topics: Amanitins; Chloroplasts; Dactinomycin; Dicarboxylic Acids; DNA, Plant; Heparin; Kinetics; Organophosphorus Compounds; Oxidoreductases Acting on Sulfur Group Donors; Pisum sativum; Rifampin; Transcription, Genetic

Previous studies in the parasitic protist Trichomonas vaginalis have revealed that protein coding genes are transcribed by an alpha-amanitin-resistant RNA polymerase (RNAP) II. To investigate whether this unusual property is a general characteristic of trichomonads, we addressed the physiology of RNA synthesis in lysolecithin-permeabilized cells. Unlike in T. vaginalis, RNAP II in Tritrichomonas foetus was highly sensitive to the inhibitor alpha-amanitin. On the other hand, RNAP III, identified by its sensitivity to the specific inhibitor tagetitoxin, was found to be resistant to alpha-amanitin in Tritrichomonas foetus, but showed a typical intermediate sensitivity in T. vaginalis. Extension of this study to an additional seven trichomonad species confirmed this genera specific pattern of alpha-amanitin sensitivity and highlighted an unusual diversity in RNAPs among trichomonads, a closely related group of unicellular eukaryotes.

Topics: Amanitins; Animals; Cell Membrane Permeability; Dicarboxylic Acids; Enzyme Inhibitors; Lysophosphatidylcholines; Organophosphorus Compounds; RNA Polymerase II; RNA, Protozoan; Transcription, Genetic; Trichomonadida; Trichomonas vaginalis; Tritrichomonas foetus

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 alpha-amanitin had no effect on gene or on viroid transcription, but tagetitoxin (5-10 microM) prevented transcription of all these genes without affecting synthesis of ASBVd strands; only at higher tagetitoxin concentrations (50-100 microM) 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.

Topics: Amanitins; Chloroplasts; Dicarboxylic Acids; DNA-Directed RNA Polymerases; Lauraceae; Nucleic Acid Hybridization; Organophosphorus Compounds; Plant Diseases; Plant Leaves; Plasmids; RNA, Catalytic; RNA, Viral; Transcription, Genetic; Viroids; Virus Replication

The HTLV-1 promoter directs RNA polymerase II transcription of viral genomic RNA in vivo. However, it has been reported that in vitro, a unique RNA polymerase, with characteristics of RNA polymerases II and III, is capable of HTLV-1 transcription (G. Piras, F. Kashanchi, M. F. Radonovich, J. F. Duvall, and J. N. Brady, J. Virol. 68:6170-6179, 1994). To further characterize the polymerase involved in HTLV-1 transcription in vitro, runoff transcription assays were performed with a variety of extracts and RNA polymerase inhibitors. Under all in vitro reaction conditions tested, RNA polymerase II appeared to be the only polymerase capable of correct transcriptional initiation from the HTLV-1 promoter. Synthesis of the specific HTLV-1 RNA transcript showed sensitivities to the RNA polymerase inhibitors tagetitoxin and alpha-amanitin that are consistent with RNA polymerase II transcription. Together, these data indicate that in vitro, as in vivo, the HTLV-1 promoter directs transcription by RNA polymerase II.

Topics: Amanitins; Cell Line; Cyclic AMP Response Element-Binding Protein; Dicarboxylic Acids; Gene Products, tax; HeLa Cells; Human T-lymphotropic virus 1; Humans; Organophosphorus Compounds; RNA Polymerase II; RNA Polymerase III; RNA, Viral; Transcription, Genetic

The spliced leader (SL) RNA plays a key role in mRNA maturation in trypanosomatid protozoa by providing the SL sequence, which is joined to the 5' end of every mRNA. As a first step towards a better understanding of the biogenesis and function of the SL RNA, we expressed a tagged SL RNA gene in a cell-free system of procyclic Trypanosoma brucei cells. Transcription initiates at + 1 can be detected as early as 1 min after addition of extract. Transcription of the SL RNA gene in vitro, as well as in permeable cells, is mediated by an alpha-amanitin/tagetitoxin resistant complex, suggesting a promoter that is intermediate between a classical RNA polymerase II and RNA polymerase III promoter. An analysis of the promoter architecture of the SL RNA gene revealed that regulatory elements are located upstream of the coding region and that the SL sequence, in contrast to the nematode SL sequence, is not required for T. brucei SL RNA gene transcription.

Topics: Amanitins; Animals; Base Sequence; Dicarboxylic Acids; Genes, Protozoan; Molecular Sequence Data; Mutation; Nucleic Acid Synthesis Inhibitors; Organophosphorus Compounds; Promoter Regions, Genetic; RNA Splicing; RNA, Messenger; RNA, Protozoan; Transcription, Genetic; Trypanosoma brucei brucei

We have characterized the crithidial RNA polymerases (RNAPs). The RNAPs from this organism were resolved by chromatography and could be classified into RNAP I, II and III because of their differential sensitivity to class-specific RNA polymerase inhibitors, such as alpha-amanitin (ama) and tagetitoxin. The three RNAP classes were subsequently characterized in nuclear run-ons using class-specific DNA templates and the inhibitory effect of ama on RNA chain elongation. These experiments showed that Crithidia fasciculata contains the normal set of eukaryotic RNAPs. However, RNAP II was found to be relatively resistant to ama, which seems to be a general feature of kinetoplastid parasites. Tagetitoxin, a potent inhibitor of purified crithidial RNAP III, does not inhibit RNAP III in nuclear run-on experiments, suggesting that the inhibitory effect of tagetitoxin depends on the enzyme conformation, i.e. 'naked' enzyme versus that in a transcription complex. Finally, the role of RNAP III in the transcription of the mini-exon genes, which provide the 5' end of each mRNA, is discussed.

Topics: Amanitins; Animals; Cell Nucleus; Chromatography, DEAE-Cellulose; Crithidia; Dicarboxylic Acids; DNA-Directed RNA Polymerases; Electrophoresis, Polyacrylamide Gel; Exons; Organophosphorus Compounds; RNA, Protozoan; Transcription, Genetic

We demonstrate that tagetitoxin, a bacterial phytotoxin, preferentially inhibits eukaryotic RNA polymerase III. We used promoter-directed transcription of cloned genes in cell-free extracts to compare tagetitoxin inhibition of RNA polymerases from diverse sources. In HeLa cell extracts, accumulation of 5 S rRNA, and U6 snRNAs (transcribed by RNA polymerase III) was inhibited at 0.3-3.0 microM tagetitoxin but transcription from adenovirus 2 major late promoter (RNA polymerase II) was not significantly affected at concentrations below 30 microM. Tagetitoxin also inhibited promoter-directed RNA polymerase III transcription in cell-free extracts from Bombyx mori (pre-tRNA), and Saccharomyces cerevisiae (pre-tRNA) at 0.3-3.0 microM, concentrations that also inhibit chloroplast or bacterial promoter-directed transcription. In nonspecific transcription assays, partially purified B. mori RNA polymerase III was inhibited by tagetitoxin at concentrations that inhibit Escherichia coli RNA polymerase; purified calf thymus RNA polymerase II was not inhibited by tagetitoxin. Using injection into Xenopus laevis oocytes, we compared tagetitoxin effects on accumulation of U1 snRNA, hH2B mRNA (transcribed by RNA polymerase II), 5 S rRNA and U6 snRNA (RNA polymerase III), and 5.8 S rRNA (RNA polymerase I). In Xenopus oocytes, RNA polymerase III transcription was preferentially inhibited by tagetitoxin.

Topics: Amanitins; Animals; Bombyx; Cattle; Dicarboxylic Acids; DNA-Directed RNA Polymerases; Escherichia coli; HeLa Cells; Histones; Humans; Oocytes; Organophosphorus Compounds; RNA Polymerase II; RNA Polymerase III; RNA, Ribosomal, 5S; RNA, Small Nuclear; Saccharomyces cerevisiae; Thymus Gland; Transcription, Genetic; Xenopus laevis