Abstract
This chapter discusses the preparation and properties of polypseudouridylic acid. Polypseudouridylic acid can be synthesized from pseudouridine diphosphate by the bacterial polynucleotide phosphorylase and from pseudouridine triphosphate by the bacterial DNA-dependent RNA polymerase. Polymerization of pseudouridine diphosphate by polynucleotide phosphorylase from Micrococcus lysodeikticus, Azotobacter vinelandii, and Escherichia coli has been accomplished on a preparative scale. Polynucleotide phosphorylase is prepared from extracts of Micrococcus lysodeikticus or Azotobacter vinelandii. The extent of polymerization is followed by measuring the orthophosphate liberated during the reaction, by the 32 P-orthophosphate-nucleoside diphosphate exchange, or paper chromatography of the reaction products. Optimal polymerization of pseudouridine diphosphate with a polynucleotide phosphorylase preparation from Micrococcus lysodeikticus was found to proceed when the oligonucleotide, pApApA, having an unesterified T-hydroxyl end group was used as primer. Pseudouridine differs from uridine in having an extra NH group for hydrogen-bonding. Accordingly, it was predicted that polypseudouridylic acid would not possess a more stable secondary structure than poly U. The ultraviolet-absorption spectrum of polypseudouridylic acid displays the alkaline bathochromic shift characteristic of pseudouridine derivatives. Polypseudouridylic acid is completely inactive in stimulating phenylalanine incorporation in the in vitro protein-synthesizing system from Escherichia coli. Under conditions where secondary structure exists to a high degree, polypseudouridylic acid is much more resistant to (1) phosphorolysis by polynucleotide phosphorylase, (2) degradation by pancreatic ribonuclease, and (3) degradation by snake venom phosphodiesterase 1° than is poly U.
Published Version
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