Nucleic Acid Templating Activity

Pretazettine (395) has been the subject of numerous biological studies, and it has been shown to exhibit a number of interesting activities (96,97,101,178-187). For example, 395 was found to inhibit HeLa cell growth as well as protein synthesis in eukaryotic cells by interfering with the peptide bond formation step (97,101). Furthermore, pretazettine inhibited the purified RNA-dependent DNA polymerase (reverse transcriptase) from avian myeloblastosis virus, a typical C-type virus (178), in an unusual fashion since it physically combined with the polymerase enzyme itself rather than interacted with the nucleic acid template. Pretazettine also exhibited antiviral activity against the Rauscher leukemia virus in mouse embryo cell cultures by suppressing viral replication (179). 

Is it possible to prepare, by a chemical reaction, a polynucleotide and mimic the synthesis of DNA Indeed, such a model can be developed from primary templates. The preformed nucleic acid (template) can then be used to direct the synthesis of complementary molecules by using the well-established concept of Watson-Crick base pairing (see Chapter 4). Orgel and his colleagues (44) have demonstrated that polyuridine [poly(U)] can act as a template in aqueous solution to help the condensation of a 5 -activated nucleotide (AMP), in the presence of a water-soluble carbodiimide via... 

Figure 4.3 The synthesis of an oligonucleotide from an activated mononucleotide, (a) Adenonine triphosphate (ATP), the substrate of enzymatic nucleic-acid synthesis. (b) An imidazolide of a nucleotide of the kind used in many non-enzymatic template-directed reactions, (c) The synthetic reaction leading to the formation of a trinucleotide. (Modified from Orgel, 2002.)...

Stereoview of the polymerase active site of HIV-1 RT [38]. The amino acid residues that compose the putative dNTP-binding site, including the three catalytically essential aspartic acids, are shown with side chains. The double-stranded nucleic acid is shown with the atomic model in the HIV-1 RT/DNA/Fab complex. The dNTP-binding site consists of structural elements from both protein and nucleic acid. The precise composition, position, and conformation of the template-primer can affect the recognition of... 

RNA replicase isolated from Qj8-infected E. coli cells catalyzes the formation of an RNA complementary to the viral RNA, in a reaction equivalent to that catalyzed by DNA-dependent RNA polymerases. New RNA strand synthesis proceeds in the 5 —>3 direction by a chemical mechanism identical to that used in all other nucleic acid synthetic reactions that require a template. RNA replicase requires RNA as its template and will not function with DNA. It lacks a separate proofreading endonuclease activity and has an error rate similar to that of RNA polymerase. Unlike the DNA and RNA polymerases, RNA replicases are specific for the RNA of their own virus the RNAs of the host cell are generally not replicated. This explains how RNA viruses are preferentially replicated in the host cell, which contains many other types of RNA. 

While peptide antibiotics are synthesized according to enzyme-controlled polymerization patterns, both proteins and nucleic acids are made by template mechanisms. Tire sequence of their monomer emits is determined by genetically encoded information. A key reaction in the formation of proteins is the transfer of activated aminoacyl groups to molecules of tRNA (Eq. 17-36). Tire tRNAs act as carriers or adapters as explained in detail in Chapter 29. Each aminoacyl-tRNA synthetase must recognize the correct tRNA and attach the correct amino acid to it. The tRNA then carries the activated amino acid to a ribosome, where it is placed, at the correct moment, in the active site. Peptidyltransferase, using a transacylation reaction, in an insertion mechanism transfers the C terminus of the growing peptide chain onto the amino group of... 

Phase I and II clinical trials indicated that acronycine reduced pain of the spine in some patients with multiple myeloma [280,282,283]. Acronycine has been reported to cause leukopoenia and to have CNS-depressant activity [284], Biochemically, acronycine inhibits incorporation of extracellular nucleosides into the RNA and DNA of leukaemia L-5178Y cell culture. There is, however, no evidence of interaction between acronycine and DNA or inhibition of template activity of DNA. This alkaloid does not inhibit nucleic acid synthesis in the cell, but rather inhibits the accumulation of extracellular uridine or thymidine, as nucleotides, in the intracellular precursor pool [285, 286], Acronycine, acting primarily on membranous organelles [287], seems to interfere with the structure, function and/or turnover of cell membrane components, thereby changing the fluidity of the plasma membrane [288]. 

Effect of Tilorone on the Template Activity of Nucleic Acids. . . 139... 

Polyketide synthases, fatty acid synthases, and non-ribosomal peptide synthetases are a structurally and mechanistically related class of enzymes that catalyze the synthesis of biopolymers in the absence of a nucleic acid or other template. These enzymes utilize the common mechanistic feature of activating monomers for condensation via covalently-bound thioesters of phosphopantetheine prosthetic groups. The information for the sequence and length of the resulting polymer appears to be encoded entirely within the responsible proteins. 

Polypeptides would have played only a limited role early in the evolution of life because their structures are not suited to self-replication in the way that nucleic acid structures are. However, polypeptides could have been included in evolutionary processes indirectly. For example, if the properties of a particular polypeptide favored the survival and replication of a class of RNA molecules, then these RNA molecules could have evolved ribozyme activities that promoted the synthesis of that polypeptide. This method of producing polypeptides with specific amino acid sequences has several limitations. First, it seems likely that only relatively short specific polypeptides could have been produced in this manner. Second, it would have been difficult to accurately link the particular amino acids in the polypeptide in a reproducible manner. Finally, a different ribozyme would have been required for each polypeptide. A critical point in evolution was reached when an apparatus for polypeptide synthesis developed that allowed the sequence of bases in an RNA molecule to directly dictate the sequence of amino acids in a polypeptide. A code evolved that established a relation between a specific sequence of three bases in RNA and an amino acid. We now call this set of three-base combinations, each encoding an amino acid, the genetic code. A decoding, or translation, system exists today as the ribosome and associated factors that are responsible for essentially all polypeptide synthesis from RNA templates in modem organisms. The essence of this mode of polypeptide synthesis is illustrated in Figure 2.8. 

As follows from Table 23 the unmodified EA-tRNA does not inhibit the DNA polymerase activity of FLV at the concentrations used in the incubation mixture. However, at the same concentrations the thiolated EA-tRNA is a very strong inhibitor of DNA-polymerase activities, endogenous as well as template-dependent, of FLV. Experiments are in progress to modify nucleic acid fractions of viral origin. 

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