A-Amanitine

One peptide toxin from the mushroom Amanita phalhides, a-amanitin, is a specific differential inhibitor of the eukaryotic nuclear DNA-dependent RNA polymerases and as such has proved to be a powerful research tool (Table 37-2). a-Amanitin blocks the translocation of RNA polymerase during transcription. 

Falchuk, K.H., B. Mazus, E. Ber, L. Ulpino-Lobb, and B.L. Vallee. 1985. Zinc deficiency and the Euglena gracilis chromatin formation of an a-amanitin-resistant RNA polymerase II. Biochemistry 24 2576-2580. 

Transcription factors (such as TFIID for RNA polymerase II) help to initiate transcription. The requirements for termination of transcription in eukaryotes are not well understood. All transcription can be inhibited by actinomycin D. In addition, RNA polymerase II is inhibited by a-amanitin (a toxin from certain mushrooms). These points are summarized in Table 1-3-1,... 

Inhibited by rifampin Actinomycin D RNAP 2 inhibited by a-amanitin (mushrooms) Actinomycin D... 

Amatoxins are a family of cyclic peptides, with oc-amanitin and 3-amanitin (Figure 3.1) accounting for >90% of the total amatoxins. In A. virosa, mushrooms collected in Virginia a-amanitin found to be completely replaced by amaninamide (Figure 3.1). The peptides are not destroyed by cooking and... 

Figure 3.1 Structures of the main amatoxins a-amanitin, (3-amanitin, and amaninamide.

Transcription is catalyzed by DNA-dependent RNA polymerases. These act in a similar way to DNA polymerases (see p. 240), except that they incorporate ribonucleotides instead of deoxyribonucleotides into the newly synthesized strand also, they do not require a primer. Eukaryotic cells contain at least three different types of RNA polymerase. RNA polymerase I synthesizes an RNA with a sedimentation coef cient (see p. 200) of 45 S, which serves as precursor for three ribosomal RNAs. The products of RNA polymerase II are hnRNAs, from which mRNAs later develop, as well as precursors for snRNAs. Finally, RNA polymerase III transcribes genes that code for tRNAs, 5S rRNA, and certain snRNAs. These precursors give rise to functional RNA molecules by a process called RNA maturation (see p. 246). Polymerases II and III are inhibited by a-amanitin, a toxin in the Amanita phalloides mushroom. 

DNA-directed RNA polymerase [EC 2.1.1.6] catalyzes the DNA-template-directed extension of the 3 -end of an RNA strand by one nucleotide at a time thus, n nucleoside triphosphate generate RNA and n pyrophosphate. The enzyme can initiate a chain de novo. Three forms of the enzyme have been distinguished in eukaryotes on the basis of sensitivity of a-amanitin and the type of RNA synthesized. See also Replicase... 

This mushroom produces the toxin, a-amanitin, a cyclic octapeptide having several modified amino acids and a central purine, which strongly binds to and inhibits RNA pal ii and thereby blocirs elongation. 

RNA pol II is essential for proper function of cells in all tissues and organs, but potentially fatal liver and kidney failure is the main risk for victims of a-amanitin poisoning. 

Molecular a-amanitin/ cyclopept./selective inhibits of eukaryotic RNA... 

The mushroom Amanita phalloides has evolved a very effective defense mechanism against predators. It produces a-amanitin, which disrupts mRNA formation in animal cells by blocking Pol II and, at higher concentrations, Pol III. Neither Pol I nor bacterial RNA polymerase is sensitive to a-amanitin—nor is the RNA polymerase II of A. phalloides itself ... 

The eukaryotic RNA polymerases are not inhibited by rifamycin, but RNA polymerases II and III are completely inhibited by the mushroom poison a-amanitin (see Box 28-B). Inhibitors of DNA gyrase (Chapter 27) also interfere with transcription as do chain terminators such as cordycepin (3 -deoxyadenosine) and related nucleosides. 

Several deadly species of the genus Amanita produce colorless toxic octapeptides, the amani-tins.a b Two residues of glycine, one of L-isoleucine, one of the unusual L-dihydroxyisoleucine, one of L-asparagine, and one of L-hydroxyproline are present in a-amanitin. In the center a modified tryptophan residue has been combined oxidatively with an SH group of a cysteine residue. If the dihy-droxyisoleucine residue of a-amanitin is replaced with unhydroxylated leucine, the resulting compound, known as amanullin, is nontoxic. The LD50 for mice is 0.3 mg kg 1 and 50 g of fresh Amanita phalloides may be sufficient to kill a person. Arnan-itins act slowly, and it is impossible to kill mice in less than 15 h, no matter how high the dose. 

Nuclear extracts can be fractionated by chromatography on DEAE-cellulose to give three peaks of RNA polymerase activity (the use of column chromatography is explained in chapter 6). These three peaks correspond to three different RNA polymerases (I, II, and III), which differ in relative amount, cellular location, type of RNA synthesized, subunit structure, response to salt and divalent cation concentrations, and sensitivity to the mushroom-derived toxin a-amanitin. The three polymerases and some of their properties are summarized in table 28.4. 

RNA polymerase I is located in the nucleolus and synthesizes a large precursor that is later processed to form rRNA. It is completely resistant to inhibition by a-amanitin. RNA polymerase II is located in the nucleoplasm and synthesizes large precursor RNAs (sometimes called heterogeneous nuclear RNA, or hnRNA) that are processed to form cytoplasmic mRNAs. It is also responsible for the synthesis of most viral RNA in virus-infected cells. PolII is very sensitive to a-amanitin, being inhibited by 50% at 0.05 /u,g/ml. RNA polymerase III is also located in the nucleoplasm and synthesizes small RNAs, such as 5S RNA and the precursors to tRNAs. This enzyme is somewhat resistant to a-amanitin, requiring about 5 /u,g/ml to reach 50% inhibition. 

The most useful inhibitor of eukaryotic transcription has been a-amanitin, a major toxic substance in the poisonous mushroom Amanita phalloides. The toxin preferentially binds to and inhibits RNA polymerase II (see table 28.4). At high concentrations it also can inhibit RNA polymerase III but not RNA polymerase I or bacterial, mitochondrial, or chloroplast RNA polymerases. 

The biochemical mode of action has been studied by several authors (16, 18). It appears that metalaxyl inhibits RNA synthesis by Interference with template-bound and a -amanitin-insensitive RNA polymerase action (15). 

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