Use of adenosine 5′-0-(2-thiotriphosphate) for revealing of newly initiated transcripts in isolated rat liver nuclei

Abstract

Initiation of DNA-dependent RNA synthesis in isolated rat liver nuclei was studied with adenosine 5'-0-(2-thiotriphosphate), (beta-S-ATP), as a precursor. The newly made RNA labelled with sulfur at 5'-triphosphate termini (thio-RNA) was isolated by affinity chromatography on a mercury-agarose column. Sulfur label can be removed from thio-RNA by digestion with phosphodiesterase I and nucleotide pyrophosphatase. Gel electrophoresis revealed that thio-RNA synthesized during 30 min was composed of 4S-35S molecules with three prevailing classes grouped around 4S-5S, 16S and approximately 35S. Differential sensitivity of the thio-RNA classes to low (1 microgram/ml) and high (200 micrograms/ml) concentrations of alpha-amanitin disclosed that beta-S-ATP was used for initiation of transcription by all three classes of RNA polymerases, and that thio-RNA included molecules as large as 18S initiated by RNA polymerase II. Thio-RNA resistant even to high doses of alpha-amanitin represents probably a product of RNA polymerase I which was initiated and elongated up to 35S.