Reverse transcriptases bacterial

The next stage is to ensure that the recombinant DNA molecule is copied by the enzymes which s)mthesize nucleic acids. These DNA and RNA polymerases synthesize an exact copy of either DNA or RNA from a pre-existing molecule. In this way the DNA polymerase duplicates the chromosome before each cell division such that each daughter cell will have a complete set of genetic instructions which are then passed to the newly formed RNA by RNA polymerase. While both DNA and RNA polymerase require a preformed DNA template, some viruses (such as HIV) have an RNA genome. To duplicate that genome, and incorporate it into a bacterial or mammalian cell, the viruses encode a reverse transcriptase enzyme which produces a DNA copy from an RNA template. 

RNA to initiate cDNA synthesis. All cellular mRNA contains multiple repeats of adenine bases (poly-A tails). Therefore the complementary thymine bases (oligo-dT) can be used as a primer that binds to the mRNA template required for the reverse transcriptase to synthesize the cDNA. In the case of pancreatic mRNAs (Figure 4.2), the signihcantly higher mRNA for insulin compared with other proteins allowed success in isolating the insulin-specihc cDNA. Subsequent insertion of cDNA into a bacterial expression vector allowed the production of functional insulin that is now marketed as a successful therapeutic product (Figure 4.2). 

Some well-characterized eukaryotic DNA transposons from sources as diverse as yeast and fruit flies have a structure very similar to that of retroviruses these are sometimes called retrotransposons (Fig. 26-33). Retro-transposons encode an enzyme homologous to the retroviral reverse transcriptase, and their coding regions are flanked by LTR sequences. They transpose from one position to another in the cellular genome by means of an RNA intermediate, using reverse transcriptase to make a DNA copy of the RNA, followed by integration of the DNA at a new site. Most transposons in eukaryotes use this mechanism for transposition, distinguishing them from bacterial transposons, which move as DNA directly from one chromosomal location to another (see Fig. 25-43). 

Fig. 26-4A) synthesized DNA normally. This finding stimulated an intensive search for new polymerases. Two were found DNA polymerases II (gene pol B)264 and III. Both are present in amounts less than 25% of that of DNA polymerase I.265 266 Both have properties similar to those of polymerase I, but there are important differences. By now DNA polymerases have been isolated from many organisms, many genes have been cloned and many sequences, both of bacterial and eukaryotic polymerases are known. Comparisons of both sequences and three-dimensional structures,266a/b a few of which are shown in Fig. 27-12, suggest that the polymerases belong to at least six families (Table 27-1). These include the RNA-dependent DNA polymerases known as reverse transcriptases as well as some RNA polymerases.267 2681... 

Bacterial Reverse Transcriptase Catalyzes Synthesis of a DNA-RNA Molecule Telomerase Facilitates Replication at the Ends of Eukaryotic Chromosomes Other Enzymes That Act on DNA... 

Bacterial Reverse Transcriptase Catalyzes Synthesis of a DNA-RNA Molecule... 

The endogenic reaction is catalyzed by the viral RNA (70s), template for the reverse transcriptase. It is not clear whether the higher activity of distamycin/Gly in this system, compared to the template activity of DNA in bacterial system (Table 10), is due to its higher affinity for viral RNA. This was investigated by using various exogenous templates in the DNA-polymerase reaction of FL-virions. 

Another improvement for tests when the initial template is RNA rather than DNA is to have a single thermostable enzyme that can function both as a reverse transcriptase and as a DNA polymerase (M12). This would greatly simplify tests for RNA viruses, for bacterial targets that utilize ribosomal RNA targets, and for cancer tests such as for the hybrid mRNA associated with the BCR-ABL chromosomal translocation in chronic myeloid leukemia. 

The mechanism of action of rifamycins involves primarily a strong, but noncovalent, interaction with DNA-dependent RNA polymerase enzyme in sensitive bacterial cells. The mammalian enzyme is not affected, which explains the selective toxicity neither is it mutated to resistant organisms. RNA polymerase has two components. The core enzyme contains polypeptide subunits a, J3, and i and a c factor, which are needed for recognition of RNA synthesis initiation sites. The drug binds to the J subunit of the complete enzyme only. The result is effective inhibition of RNA synthesis. It is of interest that many rifampinlike hydrazine derivatives were also found to be potent inhibitors of reverse transcriptase and shown to have antiviral properties. 

Unlike E. coli DNA polymerases, there is no 30 to 50 exonuclease proofreading activity in the E. coli RNA polymerase and, therefore, the error rate is relatively higher (-10-410-5). Since RNA represents only a transient copy of DNA, and is not inherited through the germ line, this error frequency is tolerable. As discussed in Chapter 30, viral RNA polymerases and another enzyme, reverse transcriptase, which also lacks proofreading activity, have error frequencies in the same range as the bacterial RNA polymerase. This mutation rate is most likely beneficial to some viruses because it results in frequent alterations in viral protein sequences and thus allows the virus to escape immune-system defenses in the host (Chapter 30). 

Retroviruses of vertebrates are, perhaps, the most widely studied class of eukaryotic transposable elements. These RNA viruses use reverse transcriptase to synthesize a circular duplex DNA, which can integrate into many sites of the host cell chromosome. The integrated retroviral genome bears remarkable resemblance to a bacterial composite transposon (compare Figure 25.38 with Figure 25.35). 

MECHANISM OF ACTION AND BACTERIAL RESISTANCE Rifampin forms a stable complex with DNA dependent RNA polymerase, leading to suppression of initiation of chain formation (but not chain elongation) in RNA synthesis. High concentrations of rifampin can inhibit RNA synthesis in mammahan mitochondria, viral DNA-dependent RNA polymerases, and reverse transcriptases. Rifampin is bactericidal for both intracellular and extracellular microorganisms. 

The DNA sequence to be inserted in the bacterial plasmid to direct the production of a-globin should be cDNA, which is a sequence complementary to the mRNAfor a-globin. The cDNA can be produced on the mRNA template in a reaction catalyzed by reverse transcriptase. 

An ansamycin antibiotic produced by Streptomyces spectabilis with antibacterial (against tuberculosis pathogens), antiviral, and antitumor activities. S. occurs as a multi-component mixture of up to ten individual compounds (S. A to G, J, K, and U) with S. C (methyl streptovaricate, C4oH5,NO,4, Mr 769.84, amorphous, mp. 189-191 °C, [a][) 4-602°) as the main component. The S. are inhibitors of bacterial RNA-polyme-rase as well as the reverse transcriptase of oncogenic viruses. The biosynthesis of the aromatic core branches off from the shikimic acid pathway while the alkyl chain is formed on the polyketide pathway. 

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