In vitro premature termination in SV40 late transcription

Abstract

Nuclear extracts and viral transcribing minichromosomes were prepared from SV40-infected cells and incubated in vitro with [alpha-32P]UTP under conditions which allow the elongation of preinitiated RNA chains. Sucrose gradient lysis of the transcription mixtures revealed two populations of SV40-specific RNA: elongating chains that remain associated with the viral minichromosomes, and, at the top of the gradient, small free RNA detached from the template and hybridizing exclusively to the promoter-proximal region of SV40 DNA. This free RNA was shown by polyacrylamide gel electrophoresis to comprise essentially a 94 nucleotide species, which could, however, at high UTP concentration, be elongated a further few nucleotides before terminating. These results thus show that the actively transcribing minichromosomes provide a sytem in which the attenuated RNA can be released from the template. Moreover, this is the first demonstration of specific in vitro termination of polymerase B transcription. The conditions which lead to transcription termination are discussed.