Factors influencing RNA degradation by Thermus thermophilus polynucleotide phosphorylase

Abstract

At the optimal temperature (65 degrees C), Thermus thermophilus polynucleotide phosphorylase (Tth PNPase), produced in Escherichia coli cells and isolated to functional homogeneity, completely destroys RNAs that possess even a very stable intramolecular secondary structure, but leaves intact RNAs whose 3' end is protected by chemical modification or by hybridization with a complementary oligonucleotide. This allows individual RNAs to be isolated from heterogeneous populations by degrading unprotected species. If oligonucleotide is hybridized to an internal RNA segment, the Tth PNPase stalls eight nucleotides downstream of that segment. This allows any arbitrary 5'-terminal fragment of RNA to be prepared with a precision similar to that of run-off transcription, but without the need for a restriction site. In contrast to the high Mg(2+) requirements of mesophilic PNPases, Tth PNPase retains significant activity when the free Mg(2+) concentration is in the micromolar range. This allows minimization of the Mg(2+)-catalysed nonenzymatic hydrolysis of RNA when phosphorolysis is performed at a high temperature. This capability of Tth PNPase for fully controlled RNA phosphorolysis could be utilized in a variety of research and practical applications.