Nucleic Acids Research, 1984, Vol. 12, No. 13 5369-5384
© 1984
MOLECULAR BIOLOGY |
Specific termination of in vitro transcription by calf thymus RNA polymerase III
Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California 405 Hilgard Avenue, Los Angeles, CA 90024, USA
Received May 16, 1984. Accepted May 31, 1984.
In vitro transcription of cleaved SV4O DNA with calf thyrnus RNA polymerase reveals a discrete transcript. The pattern of resistance to the inhibitor 
-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 SV4O 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 SV4O 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 Alul-family transcripts, a collection of SV4O polymerase III transcripts is also produced, These do not depend on restriction cleavage of the DNA and thus arise from transcription of intact DNA.