In vitro transcription and immobilized template analysis of preinitiation complexes.

In the study of gene regulation, it is often necessary to employ functional assays that investigate the action or mechanism of specific promoters or enhancer-binding factors and their role in transcription by RNA polymerase II. Although many assays measure the transcription of a gene under the control of an endogenous or model activator in vivo, it is often useful to recreate transcription in vitro in order to study specific regulatory mechanisms. In this unit, protocols are presented that will allow the investigator to perform in vitro transcription using preinitiation complexes assembled from cellular extracts on either naked DNA or chromatin templates.