Transcription activation via enhanced preinitiation complex assembly in a human cell-free system lacking TAFIIS

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.