8] Purification and analysis of functional preinitiation complexes preinitiation complexes

Abstract

Publisher Summary The initiation of transcription in eukaryotes requires the assembly at the promoter of a plethora of proteins in addition to RNA polymerase II. A more thorough analysis of transcriptional regulation requires techniques that can be used to monitor preinitiation complex assembly in a crude nuclear extract. This chapter describes methods that were developed for this purpose. These methods are linked to functional transcription assays and, therefore, have additional applications in the study of transcriptional regulation. The chapter describes two different techniques by which this can be achieved. The first method utilizes a linear DNA template immobilized on a solid support that facilitates separation of preinitiation complexes from unbound material. The second method involves the formation of transcription complexes on a supercoiled DNA template that are resolved from unbound factors by gel-filtration chromatography. The latter method is more labor intensive and is not generally appropriate for batch processing of samples or staged incubations involving more than one purification step.