Transcription chain termination

Cordycepin, or 3 -deoxyadenosine, is a transcription chain terminator because it lacks a 3 hydroxyl group from which to extend. The nucleotide of cordycepin is incorporated into growing chains, confirming that transcriptional chain growth occurs in a 5 to 3 direction. 

Figure 37-6. The predominant bacterial transcription termination signal contains an inverted, hyphenated repeat (the two boxed areas) followed by a stretch of AT base pairs (top figure). The inverted repeat, when transcribed into RNA, can generate the secondary structure in the RNA transcript shown at the bottom of the figure. Formation of this RNA hairpin causes RNA polymerase to pause and subsequently the p termination factor interacts with the paused polymerase and somehow induces chain termination.

Fludarabine is an analog of the purine adenine. It interferes with DNA polymerase to cause chain termination and inhibits transcription by its incorporation into RNA. Fludarabine is dephosphorylated rapidly and converted to 2-fluoro-Ara-AMP (2-FLAA), which enters the cells and is phosphorylated to 2-fluoro-Ara-ATP, which is cytotoxic. Fludarabine is converted rapidly to 2-FLAA. The pharmacokinetics of 2-FLAA... 

The protein truncation test is a way of testing large genes (e.g., NEl) for which an antibody is available. PTT can detect nonsense mutations that are peptide chain terminating. These show up, after reverse transcription/cell-free translation, as shorter-than-normal peptides in an electrophoretic gel (Eig. 3C). 

It is synthetic nucleoside analogue active against HIV. It is phosphorylated to its active 5 -triphosphate metabolite (L-TP). Lamivudine triphosphate inhibit HIV reverse transcription via viral DNA chain termination. 

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. 

The human immunodeficiency virus replicates by converting its single-standed RNA into double-stranded DNA which is incorporated into host DNA this crucial conversion, the reverse of the normal cellular transcription of nucleic acids, is accomplished by the enzyme reverse transcriptase. Zidovudine, as the triphosphate, was the first anti-HIV drug to be introduced and has a high affinity for reverse transcriptase. It is integrated by it into the viral DNA chain, causing premature chain termination. The drug must be present continuously to prevent viral alteration of the host DNA, which is permanent once it occurs. 

To be converted into an active compound, all the NRTIs have to be phosphorylated intracellularly to the triphosphate form via monophosphates and diphosphates. The triphosphates compete with cellular nucleotides and inhibit HIV reverse transcriptase by introducing a chain terminator into the growing complementary DNA strand during reverse transcription. However, this mechanism also occurs when human DNA is transcribed by the human DNA poljmerase. In fact, the first NRH in clinical use (zidovudine) was initially developed as an anticancer drug, targeting human DNA polymerase in cancer cells. However, aU NRTIs have higher specificity for viral reverse transcriptase than for the human DNA polymerase. 

Fludarabine monophosphate (FAMP) is an analog of the purine adenine. Like cytarabine, fludarabine interferes with DNA polymerase, causing chain termination. Unlike ara-C, fludarabine is also incorporated into RNA, resulting in inhibited transcription. The usual dose-limiting toxicity is myelosuppression. Fludarabine is also immunosuppressive, with associated opportunistic infections. ... 

The details of the chain of events in translation differ somewhat in prokaryotes and eukaryotes. Like DNA and RNA synthesis, this process has been more thoroughly studied in prokaryotes. We shall use Escherichia coli as our principal example, because aU aspects of protein synthesis have been most extensively studied in this bacterium. As was the case with replication and transcription, translation can be divided into stages—chain initiation, chain elongation, and chain termination. 

The techniques for DNA sequencing as described above can also be used, with slight modifications for the sequencing of RNA. RNA molecules must initially be transcribed to their complementary DNA (cDNA) sequence by reverse transcription (section 6.3.5). The resulting cDNA can be sequenced by either the chemical cleavage or the chain terminator method. 

Chain termination. Two types of transcription termination operate in prokaryotes ... 

Potential Effects on Transcription or Translation To complement direct polymerase intaaction assays, the potential effects of nonnatural monomeric metaboUles on transcription and translation processes should also be considered. Cell-free recombinant transcriplion/translation kits are commercially available (bacterial or mammalian origin) to enable the characterization of individual monomeric metabolites. In principle, any bad actor chemically modified nucleotide could be identified via alterations to overall RNA yield/integrity. Again, these assays could be conducted in competition-type formats (modified NTP spiked into endogenous NTP pool) mimicking relative exposure ratios in vivo. For translation, synthetic RNA templates could be synthesized that contain the chemical modification in question to see whether the presence of that modification in an RNA template alters overall protein yield. As an early hazard ID-type screen, any nucleotide modifications that behave as chain terminators or have some other inhibitory activity should be readily identified. 

H., and Kang, C. (2001) DNA sequencing and genotyping by transcriptional synthesis of chain-terminated RNA ladders and MALDI-TOF mass spectrometry. Nucleic Acids Res., 29 (3), Ell. 

Epivir is activated incraccllularly to its 5 -triphosphate derivative (Epivir-TP) (49), which is a weak inhibitor of the HIV-1 reverse transcriptase in vitro its main mode of action is via chain termination of reverse transcription (50). In vitro studies against HIV-1 clinical and laboratory strains in several cell lines have shown activity with an IC of 0.003-1.14 pM (12). In vitro activity has also been demonstrated agaimt Retrovir-resistant isolates (51), and in comparison with Retrovir, Epivir displays lower toxicity against normal and HIV-infected peripheral blood lymphocytes (12). 

At least four steps in the enzymic transcription of DNA into messenger RNA can be distinguished (1) the binding to a promoter with specific sequence by special factors referred to as T or auxiliary factors (M, and CAP), (2) formation of the first phospho-diester bond, (3) chain elongation, and (4) chain termination. Obviously, the molecular properties of the RNA polymerase must be known before the molecular interactions that occur at each of these steps can be clarified. 

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