Polynucleotide phosphorylases from thermophiles

Abstract

A purification procedure was developed for Eschericia coli polynucleotide phosphorylase, and subsequently applied to polynucleotide phosphorylases from Thermus aquaticus and Bacillus stearothermophilus. Preliminary investigations of the catalytic properties of the thermostable polynucleotide phosphorylases were carried out in the hope of effecting the facile polymerisation of modified nucleotide diphosphates which have a predominantly syn conformation. However, even at elevated temperatures, where the relative proportion of substrate molecules in the anti-conformation may be increased, the specificity of the thermostable enzymes was no broader than that reported for mesophylic enzymes. Other catalytic properties investigated were also similar to those observed using polynucleotide phosphorylases from other sources. Structural studies of the enzyme from B. stearothermophilus revealed a similar gross amino acid composition and molecular weight to the E. coli enzyme. The quaternary structure differs from other polynucleotide phosphorylases in that four apparently identical subunits were identified on polyacrylamide gel electrophoresis under denaturing conditions. The subunits have a molecular weight of 51,000 daltons. Suberimidate cross-linking experiments confirmed a tetrameric structure for the native enzyme. Partially purified polynucleotide phosphorylase from T. aquaticus had a molecular weight of more than 400,000 daltons as judged by gel filtration. Using a 3' exonuclease from Krebs ascites cells to degrade the rapidly labelled giant nuclear RNA from SV 40 transformed mouse cells, the location of virus specific sequences was investigated by hybridisation to purified SV 40 DNA. An apparent enrichment of virus sequences with increasing degradation of the RNA molecules suggests that virus sequences are absent at the 3’ end of giant nuclear RNA