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 TAFII-independent manner in HeLa nuclear extracts immunodepleted of TBP and major TAFIIs. Single-round transcription assays reveal that TAFII-independent activation is manifested at the level of productive preinitiation complex formation and that TAFIIs actually impair functional preinitiation complex assembly in a core promoter-specific manner. Furthermore, TAFIIs 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.
Oelgeschläger
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Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10021, USA.
Tao
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Y
Kang
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YK
Roeder
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RG
D002457:Cell Extracts / Q000737:chemistry / Q000494:pharmacology; D002474:Cell-Free System; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D056892:Mediator Complex; D016296:Mutagenesis / Q000502:physiology; D011392:Proline; D011401:Promoter Regions, Genetic / Q000502:physiology; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D015534:Trans-Activators / Q000235:genetics; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology*
D002457:Cell Extracts; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C070633:Gal-VP16; D056892:Mediator Complex; C093965:SRB7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C106785:TAF1 protein, S cerevisiae; D035381:TATA-Binding Protein Associated Factors; D015534:Trans-Activators; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D011392:Proline