Actinomycins termination

There is a single prokaryotic RNA polymerase that synthesizes all types of RNA in the cell. The core polymerase responsible for making the RNA molecule has the subunit structure Ojpp. A protein factor called sigma (a) is required for the initiation of transcription at a promoter. Sigma factor is released immediately after initiation of transcription. Termination of transcription sometimes requires a protein called rho (p) faaor. This enzyme is inhibited by rifampin. Actinomycin D binds to the DNA preventing transcription. 

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,... 

Like modular PKSs, peptide synthetases also epimerize some substrates and/or intermediates. For example, the starter substrate amino acid of cyclosporin A is D-Ala. Racemization of alanine is not catalyzed by an integrated subunit of cyclosporin A synthetase, but by alanine racemase. This is a separate, pyridoxal phosphate-dependent enzyme [ 193]. In contrast, Grsl and Tycl covalently activate L-Phe as a thioester and subsequently epimerize the amino acid [194]. D-Phe is the only epimer accepted as a substrate for dipeptide formation by Grs2 and Tyc2 [195, 196]. No racemization activity is detected in a pantetheine-deficient mutant of Grsl [197]. Deletion mutagenesis pointed to the requirement of the COOH-terminal part of the module for epimerizing L-Phe to D-Phe [180]. In contrast, the biosynthesis of actinomycin D, a bicyclic chromo-pentapeptide lactone (Fig. 10), involves formation of the dipeptide 6-MHA (methylanthranilic acid)-L-Thr-L-Val prior to epimerization of the L-Val exten-... 

Ester links between threonine and the terminal carboxyl of a peptide chain forming a lactone have been found in actinomycin (Bullock and Johnson, 1957), while Dekker et al. (1949) have found an imide link in the form of a pyrrolidonyl ring involving the N-terminal glutamic acid residue in a peptide isolated from algae. In teichoic acids (polymers present in... 

A number of methods for the study of apoptosis and necrosis by flow cytometry have been developed (see review by Steensma et al., 2003). Unfortunately, none of the methods are rigorously specific for apoptosis and many show poor overall specificity for cell death or cannot discriminate between the terminal stages of apoptosis and necrosis. Moreover, some of the fluorochromes used to detect apoptosis have emission spectra that fully overlap the spectra of those typically used for immunophenotyping or have a broad emission spectrum that makes compensation in multi- or polychromatic flow cytometry difficult, if not impossible. Thus, the preferred method would be one that measures two aspects of apoptosis, can discriminate between apoptotic and dead cells, and uses fluorochromes that allow simultaneous immunophenotyping. One such method is the annexin-V/7-amino-actinomycin-D assay described by Merchant et al. (2001) that is available in kit from BD Biosystems (San... 

Actinomycin D (Figure 26.4), is a transcriptional terminator that acts by binding to DNA. The tricyclic ring system (phenoxazone) intercalates between adjacent G-C base pairs, and the cyclic polypeptide arms fill the nearby narrow groove. 

Dactinomycin is administered by intravenous injection. The drug is excreted both in bile and in the urine and disappears from plasma with a terminal t j of 36 hours. Metabolism of the drug is minimal. Dactinomycin does not cross the blood—brain barrier. The usual daily dose of dactinomycin (actinomycin D cosmegen) is 10-15 pg/kg this is given intravenously for 5 days. If no manifestations of toxicity are encountered, additional courses may be given at intervals of 2—4 weeks. 

Alkaline hydrolysates of mRNA from vaccinia virus (Kates, 1970) and mouse sarcoma 180 cells (Mendecki et al, 1972) show that the poly (A) tracts, 100-200 nucleotides in length, are located at the 3 -ter-minal end of the RNA molecules. Analysis of the reaction products of highly purified exoribonuclease specific for 3 -OH termini also indicates that most (and possibly all) of the poly (A) sequences are at the 3 -terminal end of the mRNA s (Molloy et al, 1972). This is supported by the fact that the time course of poly( A) labeling in polysomes indicates that this sequence is assembled after the rest of the RNA molecule has been completed (Mendecki et al, 1972). Poly (A) synthesis is sensitive to actinomycin D but to an extent less than that of the rest of the RNA molecule which further argues that the poly (A) segment is added after transcription is completed (Darnell et al, 1971b Mendecki et al, 1972). 

Rifampicin and other members of the rifamycin group bind to the subunit of bacterial RNA polymerase and block the formation of the first phosphodiester bond. They therefore inhibit initiation, with little effect on elongation or termination of previously initiated chains. Actinomycin D binds tightly to double-stranded DNA and prevents it from acting as a template for transcription. It binds between adjacent base pairs, particularly in G-rich regions, by a process known as intercalation. At low concentration, RNA synthesis is effectively inhibited with little effect on DNA or protein synthesis. 

It was shown (Wicks et al., 1965 Greenman et al., 1965) that steroid hormones stimulate synthesis of all fractions of RNA including the soluble. In the last case it is synthesis of sRNA which is stimulated and not exchai e of the terminal group. However, this general stimulation is evidently a late effect. Analysis of the action of estrogen on the uterus after shorter periods of time showed (Notides and Gorski, 1966) that after 30 min synthesis of one particular protein is induced, and that this stimulation is insensitive to actinomycin D (the translation level). General stimulation of RNA and protein synthesis developed later. 

When second-strand DNA is synthesized by the one-tube method (see below), first-strand cDNA synthesis is performed without actinomycin D and the first reaction mixture is directly adjusted with the reaction buffer for second-strand synthesis. When second-strand DNA is synthesized by the conventional procedures (see AMV RTase), the first reaction is terminated by adding 2 /zl of SDS (10%, w/v) and 2 ju.1 EDTA (0.5 M, pH 8.0), and the cDNA products are precipitated with ethanol. 

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