Isolation of stable preinitiation, initiation, and elongation complexes from RNA polymerase II-directed transcription.

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RESUMO

Distinct RNA polymerase II transcription preinitiation, initiation, and elongation complexes can be formed in vitro on cloned adenovirus 2 DNA sequences containing the major late promoter. These transcription complexes are stable and can be rapidly isolated by gel filtration of HeLa whole cell extracts. In the absence of exogenous nucleotides and under appropriate salt conditions, a stable but transcriptionally incomplete preinitiation complex is formed. When this complex is incubated in the presence of adenosine or deoxyadenosine triphosphates, the beta-gamma phosphodiester bond is hydrolyzed, and RNA polymerase II joins the complex, thereby converting it into a stable initiation complex capable of forming (but prior to the formation of) the first phosphodiester bond. When this complex is isolated and incubated in the presence of all four nucleoside triphosphates, it is converted into an elongation complex that then permits the synthesis of phosphodiester bonds and the correct run-off transcript. A limiting transcription component is sequestered in the preinitiation complex. This factor is released upon elongation and can reassociate with new DNA templates during subsequent rounds of initiation. Therefore, class II genes do not appear to form activated transcription units stable for multiple rounds of transcription; rather, their transcriptional activity may be controlled in part by regulating the association of transcription factors at each initiation event.

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