Formation of a single phosphodiester bond by RNA polymerase B from calf thymus is not inhibited by alpha-amanitin.

Abstract

The template-directed synthesis of a single phosphodiester bond by highly purified calf thymus RNA polymerase B is not inhibited by high concentrations of alpha-amanitin (10(-6) M). However, a subsequent internucleotide bond is not synthesized in the presence of alpha-amanitin. These results suggest that translocation of the nascent RNA and RNA polymerase B along the DNA template is the enzymatic process inhibited by alpha-amanitin. It is also shown that the formation of a single phosphodiester bond by RNA polymerase B results in a stable ternary transcription complex, i.e., between the enzyme, the DNA, and the nascent RNA. Under reaction conditions which normally favor the elongation of RNA, the transcriptional process is arrested at initiation by alpha-amanitin. Such ternary initiation complexes have been isolated by agarose gel electrophoresis.