Mammalian RNA polymerase

The mode of action of the naphthoquinoid ansamacroHdes was estabHshed through studies using the tifamycins and streptovaricins (84,141,257,258). The ansamacroHdes inhibit bacterial growth by inhibiting RNA synthesis. This is accompHshed by forming a tight complex with DNA-dependent RNA polymerase. This complex is between the ansamacroHde and the P-unit of RNA polymerase. The formation of the complex inhibits the initation step of RNA synthesis (259,260). The ansamacroHdes form no such complex with mammalian RNA polymerase and thus have low mammalian toxicity. 

Baskaran, R. Escobar, S.R. Wang, J.Y. Nuclear c-Abl is a COOH-terminal repeated domain (CTD)-tyrosine (CTD)-tyrosine kinase-specific for the mammalian RNA polymerase II possible role in transcription elongation. Cell Growth Differ., 10, 387-396 (1999)... 

Rifampicin was first shown by Hartmann et al. 54 to have a specific inhibitory effect on RNA polymerase from E. coli. Later, other active ansamycins were found and RNA polymerases from a large variety of bacteria other than E. coli proved to be sensitive to the drug. More recently, an RNA polymerase from E. coli containing only one subunit and probably involved in the initiation of DNA replication (dna G gene product) has been shown to be resistant to rifampicin5 s This holds true also for the various mammalian RNA polymerases. In contrast to non-specific inhibitors of transcription such as actinomycin and mitomycin, rifampicin interacts specifically with the bacterial enzyme itself. With the aid of 14C-labelled rifampicin it could be shown that the drug forms a very stable complex with the enzyme in a molar ratio of 1 1S6> 57 The dissociation constant of this complex is 10-9 M at 37 °C and... 

Pohl, T, Waldmann, H, Chemoenzymatic synthesis of a characteristic phosphorylated and glycosylated peptide fragment of the large suhunit of mammalian RNA polymerase II, J. Am. Chem. Soc., 119, 6702-6710, 1997. 

Rifampicin (Fig. 10.70) is a semisynthetic rifamycin made from rifamycin B—an antibiotic isolated from Streptomyces mediterranei. It inhibits Gram-positive bacteria and works by binding non-covalently to RNA polymerase and inhibiting RNA synthesis. The DNA-dependent RNA polymerases in eukaryotic cells are unaffected, since the drug binds to a peptide chain not present in the mammalian RNA polymerase. It is therefore highly selective. 

The selectivity of this antibiotic is interesting since both bacterial cells and mammalian cells contain the enzyme RNA polymerase. However, as we have seen, the enzyme in bacterial cells contains a peptide chain not present in mammalian RNA polymerase. 

Tomaletti, S., Patrick, S.M., Turchi, and Hanawalt, P.C. (2003) Behavior of T7 RNA polymerase and mammalian RNA polymerase II at site-specific cisplatin adducts in the template DNA. J. Biol. Chem., 278, 35791-35797. 

Kuraoka, I., Endou, M., Yamaguchi, Y., Wada, T., Handa, H., and Tanaka, K. (2003) Effects of endogenous DNA base lesions on transcription elongation by mammalian RNA polymerase II. Implications for transcription-coupled DNA repair and transcriptional mutagenesis. J. Biol. Chem., 278, 7294-7299. 

Transient Transfection The simplest of the two expression methods, called transient transfection, employs a vector sIm liar to the yeast shuttle vectors described previously. For use In mammalian cells, plasmid vectors are engineered also to carry an origin of replication derived from a virus that Infects mammalian cells, a strong promoter recognized by mammalian RNA polymerase, and the cloned cDNA encoding the protein to be expressed adjacent to the promoter (Figure... 

Amanda Tin. The toxin a-amanitin is capable of causing irreversible ) hepatocellular and renal dysfunction through inhibition of mammalian RNA polymerases. Fortunately, Amanda Tin s toxicity proved mild. She developed only gastrointestinal symptoms and slight changes in her hepatic and renal function, which returned to normal within a few weeks. Treatment was primarily supportive, with fluid and electrolyte replacement for that lost through the gastrointestinal tract. No effective antidote is available for the A. phalloides toxin. 

Several of the toxic cyclopeptides of the toadstool Amanita phalloides have been shown to inhibit mammalian RNA-polymerase. ... 

Killeen, M., Coulombe, B., and Greenblatt, J. (1992). Recombinant TBP, transcription factor IIB, and RAP30 are sufficient for promoter recognition by mammalian RNA polymerase II. J. Biol. Chem. 267(14), 9463-9466. 

Ossipow, V., Iassan, J.-P., Nigg, E. A., and Schibler, U. (1995). A mammalian RNA polymerase II holoenzyme containing all components required for promoter-specific transcription initiation. Cell 83, 137-146. 

One of the fractions required to direct rDNA transcription contains the RNA polymerase I enzymatic activity. Purification of mammalian RNA polymerase I indicates that the core enzyme is a multisubunit complex with a molecular mass of >500 kDa. (Hannan et aL, 1998c Song et al.,... 

The group of rifamycin antibiotics suffered from the disadvantage of being inactive by oral administration. Chemical modification of rifamycin-SV led to rifampicin (LXXIII) which had the required oral activity [270]. Biochemical studies proved that its antibacterial action was due to inhibition of bacterial DNA-dependent RNA polymerase. It had no effect on mammalian RNA polymerase. Some DNA viruses also produce a DNA-dependent RNA polymerase and rifampicin was shown to have slight activity at 50 pg/ml and marked activity at 100 pg/ml against vaccinia virus. It began to show toxic effects in cells at 500 jig/ml [271]. 

The rifamycins act on the sensitive bacteria, blocking the synthesis of all cellular RNA, because they are potent inhibitors of all bacterial DNA-directed RNA polymerases. Most rifamycins are not effective on the mammalian RNA polymerase. Therefore, they possess the necessary requisite for ideal chemotherapeutic agents, that is, selective toxicity against pathogens. Detailed studies on the mechanism of action of rifamycins revealed that they inhibit the initiation of RNA synthesis by inactivating the enzyme before the incorporation of the first purine nucleotide of the RNA chain. Rifamycins form a rather stable equimolecular complex with the bacterial RNA polymerase, binding with the subunit of the enzyme [201-203]. 

Rutter and his associate [186, 187] have solubilized mammalian RNA polymerase by sonication of nuclear preparations in high salt followed by dilution of the suspension. After dilution, most free proteins aggregate with the DNA, but the polymerase is removed in solution. The enzyme is then purified more extensively, and as a result three chromatographic peaks are separated. The peaks can be distinguished by their response to various salt concentrations, activity on various primers, requirements for manganese and magnesium, and the effects of inhibitors. 

Dun dr M, Hoffmann-Rohrer U, Hu Q, Grummt I, Rothblum LI, Pharr RD, Misteli T (2002) A kinetic framework for a mammalian RNA polymerase in vivo. Science 298 1623-1626... 

Chemical modification of enzymes is frequently correlated with changes in levels of activity. Mammalian RNA polymerase II subunits are phosphorylated in vivo (Bell et al., 1977 Dahmus, 1981a). Labeling of HeLa cells with results in phosphate incorporation into pol II polypeptides of 240,000, 214,000, and 20,500 daltons (Dahmus, 1981a). Purified pol II is a substrate for both casein kinase I and II and for the cyclic AMP independent nuclear protein kinase NIL The... 

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