Specific termination of in vitro transcription by calf thymus RNA polymerase III

In vitro transcription of cleaved SV40 DNA with calf thymus RNA polymerase reveals a discrete transcript. The pattern of resistance to the inhibitor alpha-amanitin identifies the RNA as a product of RNA polymerase III transcription. The RNA is shown to initiate artificially near a DNA terminus created by cleavage and to terminate specifically near a cluster of 8 thymidine residues within the SV40 control region. Faithfully initiated transcripts cannot be detected using the calf thymus enzyme, supporting the idea that polymerase III termination can be accomplished by an initiation-deficient enzyme. Transcription of SV40 DNA in a HeLa cell lysate also leads to specific polymerase III transcription. When PvuII-cleaved DNA is the template, the same RNA is produced as with the calf thymus enzyme. At the lowered lysate concentration known to activate certain AluI-family transcripts, a collection of SV40 polymerase III transcripts is also produced. These do not depend on restriction cleavage of the DNA and thus arise from transcription of intact DNA.