Nucleic Acids Research, 1983, Vol. 11, No. 17 6041-6064
© 1983
MOLECULAR BIOLOGY |
RNA polymerase II ternary transcription complexes generated in vitro
The Wistar Institute of Anatomy and Biology Philadelphia, PA 19104, USA
Received April 21, 1983. Revised August 3, 1983. Accepted August 3, 1983.
Ternary transcription complexes have been formed with a HeLa cell extract, a specific DNA template, and nucleoside tr1phosphates. The assay depends on the formation of sarkosyl-resistant Initiation complexes which contain RNA polymerase II, template DNA, and radioactive nucleoside triphosphates. Separation from the other elements 1n the in vitro reaction 1s achieved by electrophoresis 1n agarose – 0.25% sarkosyl gels. The mobility of the ternary complexes in this system cannot be distinguished from naked DNA. Formation of this complex 1s dependent on all parameters necessary for faithful in vitro transcription. Complexes are formed with both the plasmid vector and the specific adenovirus DNA Insert containing a eucaryotic promoter. The formation of the complex on the eucaryotic DNA 1s sequence-dependent. An undecaribonucleotide predicted from the template DNA sequence remains associated with the DNA in the ternary complex and can be Isolated 1f the chain terminator 3'-0-methyl GTP 1s used, or after Tl ribonuclease treatment of the RNA, or if exogenous GTP 1s omitted from the in vitro reaction. This oligonucleotide is not detected in association with the plasmid vector. Phosphocellulose fractionation of the extract Indicates that at least one of the column fractions required for faithful runoff transcription 1s required for complex formation. A large molar excess of abortive Initiation events was detected relative to the level of productive transcription events, indicating a 40-fold higher efficiency of transcription initiation vs. elongation.
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