Properties of in vitro trarscription by isolated Xenopus oocyte nucleoli

Abstract

Some properties of in vitro transcription by isolated Xenopus oocyte nucleoli were described. When incubated with labeled RNA precursors, Xenopus oocyte nucleoli exhibited prolonged incorporation of radioactivity into RNA. The synthetic activity was exclusively due to type I RNA polymerase as revealed by its insensitivity to low and high doses of alpha-amanitin. The size of the in vitro transcript was mostly larger than 28S at 10 minute incubation and became smaller as incubation proceeded. When [gamma-32P]ATP was included in the reaction mixture, 32P radioactivity was incorporated into RNA suggesting the possible initiation of transcription in this system. However, analysis of the terminal nucleotide of the transcript revealed that the incorporation of radioactivity from [gamma-32P]ATP was not due to the initiation of transcription but due to polynucleotide kinase activity in the nucleolar preparation. These results demonstrate that the incorporation of radioactivity from [gamma-32P] labeled nucleoside triphosphates cannot necessarily be regarded as an index of the initiation of transcription.

A comparison of nucleoside (β-S) triphosphates and nucleoside (γ-S) triphosphates as suitable substrates for measuring transcription initiation in preparations of cell nuclei

“…The results of this experiment are consistent with our hypothesis of thiophosphate transfer by a kinase type mechanism, in which only the y-phosphate of the nucleoside triphosphate substrate is directly accessible for transfer. Oligonucleotide kinase activities have also been reported in other mammalian cell extracts (15)(16). As an alternative explanation for the binding of [32P]RNA to Hg-Sepharose in our experiments, it seems extremely unlikely that 3 to 5% of the [32P]RNA could be degraded to mononucleotides, converted to [a-32P]triphosphates, and incorporated into new thiol-containing RNA chains in the cell free reaction.…”