Abstract
An important property of RNA polymerase II preinitiation complexes (PIC) is their ability to coordinate transcription initiation with chromatin modification and remodeling. We employed multidimensional protein identification technology (MuDPIT) to study the composition and assembly of PICs in HeLa and mouse embryonic stem (ES) cell nuclear extracts. Strikingly, most of the machinery necessary for transcription initiation on chromatin is part of the PIC. The PIC is nearly identical between ES and HeLa cells, and contains two major co‐activator complexes, Mediator and SAGA. Genomewide analysis of Mediator reveals a close correlation with Pol II, TBP and mRNA levels implying a major role in PIC assembly. Mechanistic analysis of the two co‐activators showed that they play distinct roles in PIC function. Mediator coordinates assembly of the Pol II initiation factors and chromatin machinery into a PIC, while SAGA acts after PIC assembly to allow transcription on chromatin. MuDPIT analysis of Mediator isolated from native chromatin, under conditions where the transcriptional machinery remains intact, suggests that PICs assembled in vitro and in vivo are similar.
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