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J. Biol. Chem., Vol. 256, Issue 11, 5866-5873, 06, 1981

In vitro transcription by wheat germ RNA polymerase II. Initiation of RNA synthesis on relaxed, closed circular template

WS Dynan and RR Burgess

We have developed a novel method for studying the ability to wheat germ RNA polymerase II to initiate transcription in vitro on unnicked duplex DNA template. Our template is a plasmid which has been relaxed in vitro with wheat germ DNA topoisomerase I. The plasmid consists of SV40 cloned in pBR322. We transcribe briefly to synthesize highly labeled nascent RNA, then fractionate the resulting ternary complexes on an agarose gel to separate relaxed, closed circular DNA from other DNA species contaminating the template preparation. Under optimal conditions, we recover about half of the total RNA synthesized in complex with relaxed, closed circular DNA. The remaining RNA is in association with other DNA species or is free in solution. The principal conclusions we draw from these studies are: 1) relaxed, closed circular DNA can serve as a template for in vitro transcription, although the relative efficiencies of transcription are such that even a few per cent contamination with nicked or supercoiled template can cause transcription from these forms to dominate the reaction. 2) Initiation occurs on approximately 1 in 25 cloned circular molecules in the course of a 5-min reaction, under optimal conditions. This transcription is not predominantly from SV40 in vivo promoters and is either random or from a sufficient number of sites as to appear random. 3) There is a striking dependence of initiation frequency on purine nucleoside triphosphate concentration, especially with GTP.
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