RNA polymerase a-amanitin

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. 

RNA polymerase II is a eukaryotic RNA polymerase that transcribes messenger RNAs (mRNAs). Of the three eukaryotic RNA polymerases, RNA polymerase II is the most sensitive to oi-amanitin (Figure 26.4b). 

Bushnell D, Cramer P, Komberg R (2002) Structural basis of transcription amanitin-RNA polymerase II cocrystal at 2.8 A resolution. Proc Natl Acad Sci USA 99 1218... 

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,... 

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... 

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. 

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. 

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). 

RNA polymerase II transcribes messenger RNA and a few other small cellular RNAs. Class II promoters are usually defined by their sensitivity to a-amanitin. Like prokaryotic promoters, many class II promoters contain two conserved sequences, called the CAAT and TATA boxes. The TATA box is bound by a specialized transcription factor called TBP (for TATA-Binding-Factor). Binding of TBP is required for transcription, but other proteins are required to bind to the upstream (and potentially downstream) sequences that are specific to each gene. Like prokaryotic transcripts, eukaryotic RNAs are initiated with a nucleoside triphosphate. Termination of eukaryotic mRNA transcription is less well understood than is termination of prokaryotic transcription, because the 3 ends of eukaryotic mRNAs are derived by processing. See Figure 12-9. 

Eukaryotic cells contain at least four different DNA-dependent RNA polymerases. Their localization, cellular transcripts, and susceptibility to the cyclic octapeptide a-amanitin (derived from poisonous mushrooms) are shown in Table 11.3. a-Amanitin blocks the elongation phase of RNA synthesis. Although the structures of these enzymes are much more complex than that of the prokaryotic RNA polymerase, the basic mechanism is very similar to that of the prokaryotic enzyme. 

No. Eukaryotic RNA polymerases have been isolated from many tissues, and in all cases, three distinct enzymes have been found in the nucleus. All contain a number of polypeptide subunits and are complex in structure, RNA polymerase I is known to be involved specifically in the transcription of rRNA genes. RNA polymerase II gives rise to transcripts that are subsequently processed to yield mRNA. RNA polymerase 111 is responsible for the transcription of the tRNA genes and a small ribosomal RNA gene that yields a species called 55 RNA. The three polymerases are distinguishable from one another by their differential sensitivity to the drug a-amanitin (the toxic principle of the mushroom Amanita phalloides), which does not affect bacterial RNA polymerase. RNA polymerase... 

II is very sensitive to a-amanitin, while RNA polymerase I is completely resistant. RNA polymerase... 

III is moderately sensitive to this inhibitor. Mitochondria have yet another type of RNA polymerase, which is unaffected by a-amanitin but is sensitive to drugs that inhibit bacterial RNA polymerase. 

Yes. Examples are rifampicin and streptolydigin, which bind only to bacterial RNA polymerase and block its action a-amanitin binds only to eukaryotic RNA polymerase II and, to a lesser extent, to RNA polymerase III to block their actions. 

No. The most abundant RNA component of a cell is always rRNA (Chap. 7). The rRNA genes are transcribed by RNA polymerase I, which is resistant to a-amanitin. 

Is it possible that a-amanitin exerts its inhibitory effect on certain eukaryotic RNA polymerases by interfering with the availability of the substrates ... 

No. All RNA polymerases use the same substrates, the a-amanitin inhibits some RNA polymerases but not others. 

S, and 28S ribosomal RNA (Section 29.3.1). The other ribosomal RNA molecule (5S rRNA, Section 29.3.1) and all the transfer RNA molecules (Section 29.1.2) are synthesized by RNA polymerase III, which is located in the nucleoplasm rather than in nucleoli. RNA polymerase II, which also is located in the nucleoplasm, synthesizes the precursors of messenger RNA as well as several small RNA molecules, such as those of the splicing apparatus (Section 28.3.5). Although all eukaryotic RNA polymerases are homologous to one another and to prokaryotic RNA polymerase, RNA polymerase II contains a unique carboxyl-terminal domain on the 220-kd subunit this domain is unusual because it contains multiple repeats of a YSPTSPS consensus sequence. The activities of RNA polymerase II are regulated by phosphorylation on the serine and threonine residues of the carboxyl-terminal domain. Another major distinction among the polymerases lies in their responses to the toxin a -amanitin, a cyclic octapeptide that contains several modified... 

Figure 28.16. RNA Polymerase Poison. Amanita phalloides, a poisonous mushroom that produces a-amanitin. [After G. Lincoff and D. H. Mitchel, Toxic and Hallucinogenic Mushroom Poisoning (Van Nostrand Reinhold, 1977), p. 30.]...

The mode of action of mushroom-produced mycotoxins varies considerably. Alpha amanitin, amatoxin produced by some species of Amanita, is a class A poison that acts by inhibiting a critical nuclear polymerase that enables the cell to make protein. Once the function of this RNA polymerase is curtailed, basic life processes cease. Attempts to kill alpha amanitin with antibodies have proven to be even more harmful to patients than the poison itself. Most forms of mushroom poisoning can be treated with rapid lavage (induced vomiting) or medically approved ingestion of charcoal to absorb the toxin before it is absorbed into the stomach. 

Eukaryotes have four different RNA polymerases (RNA pol). Three are required for transcription of nuclear genes and the fourth for transcription of mitochondrial genes (Fig. 11.6). RNA polymerase I transcribes ribosomal RNA (rRNA), pol II transcribes mRNA and pol III tRNA and several small RNA s. The three polymerases consist of ten or more subunits. All have two large subunits with homology to the P and p subunits of the prokaryotic RNA polymerase. The three eukaryotic polymerases can be distinguished based on their sensitivity to a-amanitin, a toxin found in some types of mushrooms. RNA pol II activity is severely inhibited, pol III weakly and pol I is insensitive. The antibiotic rifampicin inhibits prokaryotic RNA polymerases. 

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