Polyribonucleotide synthesis

In addition to the cellular enzyme(s) that catalyzes DNA-directed RNA synthesis, cellular enzymes are involved in polyribonucleotide synthesis that do not use a template. Some of the properties of these enzymes are summarized in table 28.5. We have already mentioned polynucleotide phosphorylase in this chapter, and in chapter 26 we discussed the importance of DNA primase to DNA synthesis. 

Shaw, J.G. Enzymic degradation of adenosine triphosphate to adenine by tobacco leaf extracts and its relationship to measurements of polyribonucleotide synthesis Arch. Biochem. Biophys. 109 (1965) 627-633. 

For example, a functional 17-bp gene for the 53-residue human epidermal growth factor was synthesized by joining ten oligonucleotides of lengths 11-59 bp.575 DNA is often synthesized enzymatically using methods described in Chapter 26. Cloned sequences of synthetic DNA can also be transcribed to produce polyribonucleotides of any desired sequence.583 New nonenzymatic methods for RNA synthesis have also been devised.583-587... 

The first enzyme discovered that could catalyze polynucleotide synthesis was a bacterial enzyme called polynucleotide phosphorylase. This enzyme, isolated by Severo Ochoa and Marianne Grunberg-Manago in 1955, could make long chains of 5 -3 -linked polyribonucleotides starting from nucleoside diphosphates. However, there was no template requirement for this synthesis, and the sequence was uncontrollable except in a crude way by adjusting the relative concentrations of different nucleotides in the starting materials. 

Nirenberg, M. W., and J. H. Mattaei, The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc. Natl. Acad. Sci. 

Manganese and cadmium are both carcinogens and at concentrations of 10 moll induce changes in synthetic polyribonucleotides indicative of base mis-pairing, while Mg " and show no effects. 5-Mercuriuridine triphosphate has been used as a probe of in vitro transcription studies, thiol affinity chromatography being used to separate the in vitro synthesized RNA from the cellular RNA. Zn, Cd, and Hg have been found to stimulate RNA and DNA synthesis in chick embryo cultures deprived of serum the stimulation appeared to represent a non-specific event. ... 

Spectinomycin selectively inhibits protein synthesis in Gram-negative bacteria. The antibiotic binds to and acts on the 30S ribosomal subunit (see also Figure 75). Its action has similarities to that of the aminoglycosides however, spectinomycin is not bactericidal and does not cause misreading of polyribonucleotides. A high degree of bacterial resistance may develop as a result of mutation. 

Equilibrium for the reaction catalyzed by polynucleotide phosphorylase lies toward the direction of RNA degradation rather than synthesis. High concentrations of ribonucleo-side diphosphates are required to achieve the net synthesis of RNA and it is likely that their concentrations in the cell are not sufficient to drive net polynucleotide synthesis. Also, polynucleotide phosphorylase does not use a template, so the polyribonucleotides it synthesizes contain random sequences, which makes them of no value for protein synthesis. The cell uses polynucleotide phosphorylase as a degradative enzyme in conjunction with other nucleases that regulate the lifetimes of RNA molecules, including mRNA. In bacteria mRNA lifetimes are relatively short. 

Similar experiments were carried out with different synthetic polyribonucleotides. It was found, for example, that polyadenylic acid (poly A) leads to the synthesis of polylysine, and that polycytidylic acid (poly C) leads to the synthesis of polyproline. By 1964, all 64 codons had been deciphered (Table 20.4). Nirenberg, R. W. Holley, and H. G. Khorana shared the 1968 Nobel Prize in Physiology or Medicine for their seminal work. 

Polynucleotide phosphorylase an enzyme catalysing the synthesis in vitro of polyribonucleotides. 5 -Nucleoside diphosphates are added to oligonucleotide starter molecules (primers) with the release of phosphate. The resulting sequence depends on the availability of components for the reaction. Thus it is possible to synthesize homopolymers (poly A, poly U, etc.) or Copolymers (see) (e.g. poly AU). The function of P.p. in the cell is not clear. Since it also acts as an exonuclease and catalyses the reverse reaction, it is possible that the enzyme functions in the synthesis and remobilization of storage polynucleotides. 

Synthesis of Polyribonucleotides. The synthesis of large polymers of ribose nucleotides is catalyzed by an enzyme named polynucleotide phosphorylase. This enzyme was first found in extracts of Azotobacter vinelandii, and similar preparations have been obtained in other bacteria. This type of enzyme has not yet been identified in animal tissues. 

There is little information about the details of the translocation mechanism. A continuous polyribonucleotide chain is not essential, as translocation can occur with individual trinucleotides. It seems likely that movement of the mRNA is dependent on and tightly coupled to that of the tRNA with the binding sites for the tRNA providing the precision for movement by exactly one codon. Presumably, binding of EF-G and GTP after release of EF-Tu-GDP following peptide bond synthesis induces a conformational change in the ribosome which leads to translocation. 

The mode of action of streptomycin was studied with subcellular systems for protein synthesis, programmed by synthetic polyribonucleotides, or by phagic messenger RNAs. 

Rosemond-Hornbeak, H., and Moss, B., 1975, Inhibition of host protein synthesis by vaccinia virus, fate of cell mRNA and synthesis of small poly(A)-rich polyribonucleotides in the presence of actinomycin-D, J, ViroL 16 34. 

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