Transcription Activation via Enhanced Preinitiation Complex Assembly in a Human Cell-Free System Lacking TAFIIs

Abstract In contrast to previous findings in cell-free systems reconstituted with partially purified metazoan factors, we demonstrate dramatic activation of transcription in a TBP-dependent but TAF II -independent manner in HeLa nuclear extracts immunodepleted of TBP and major TAF II s. Single-round transcription assays reveal that TAF II -independent activation is manifested at the level of productive preinitiation complex formation and that TAF II s actually impair functional preinitiation complex assembly in a core promoter–specific manner. Furthermore, TAF II s appear to elevate absolute levels of transcription under multiple-round transcription conditions, presumably by facilitating secondary initiation events. Finally, human coactivator activities related to those in yeast RNA polymerase II/mediator complexes appear to function in unfractionated HeLa nuclear extracts.