Activator-independent functions of the yeast mediator sin4 complex in preinitiation complex formation and transcription reinitiation.
RNA polymerase II (Pol II) Mediator plays an essential role in both basal and activated transcription. Previously, subunits of the Sin4 Mediator complex (Sin4, Pgd1, Gal11, and Med2) have been implicated in both positive and negative transcriptional regulation. Furthermore, it was proposed that this subcomplex constitutes an activator-binding domain. A yeast nuclear-extract system was used to investigate the biochemical role of the Sin4 complex. In contrast to previous findings, we found at least two general activator-independent roles for the Sin4 complex. First, mutations in sin4 and pgd1 destabilized the Pol II-Med complex, leading to a reduced rate and extent of preinitiation complex (PIC) formation both in the presence and absence of activators. Although reduced in amount compared with the wild type, PICs that are formed lacking the Sin4 complex are stable and can initiate transcription normally. Second, mutation of pgd1 causes partial disruption of the Sin4 complex and leads to a defect in transcription reinitiation. This defect is caused by dissociation of mutant Mediator from promoters after initiation, leading to nonfunctional Scaffold complexes. These results show that function of the Sin4 complex is not essential for transcription activation in a crude in vitro system but that it plays key roles in the general transcription mechanism.
Reeves
Wendy M
WM
Molecular and Cellular Biology Program, University of Washington, Seattle 98105, USA.
Hahn
Steven
S
D002457:Cell Extracts; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D056892:Mediator Complex; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D035381:TATA-Binding Protein Associated Factors; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D035362:Transcription Factor TFIID / Q000235:genetics / Q000378:metabolism; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D019473:Transcription Factors, TFII / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D002457:Cell Extracts; D005656:Fungal Proteins; C470560:MED2 protein, S cerevisiae; D046911:Macromolecular Substances; D056892:Mediator Complex; D009687:Nuclear Proteins; C110281:PGD1 protein, S cerevisiae; C076878:SIN4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C490039:TAF6 protein, S cerevisiae; D035381:TATA-Binding Protein Associated Factors; D015534:Trans-Activators; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C066644:transcription factor TFIIE; D051758:Transcription Factor TFIIH; D012319:RNA Polymerase II