Abstract
The gene encoding ribonucleotide reductase 3 (RNR3) is strongly induced in response to DNA damage. Its expression is strictly dependent upon the TAF(II) subunits of TFIID, which are required for the recruitment of SWI/SNF and nucleosome remodeling. However, full activation of RNR3 also requires GCN5, the catalytic subunit of the SAGA histone acetyltransferase complex. Thus, RNR3 is dependent upon both TFIID and SAGA, two complexes that deliver TATA-binding protein (TBP) to promoters. Furthermore, unlike the majority of TFIID-dominated genes, RNR3 contains a consensus TATA-box, a feature of SAGA-regulated core promoters. Although a large fraction of the genome can be characterized as either TFIID- or SAGA-dominant, it is expected that many genes utilize both. The mechanism of activation and the relative contributions of SAGA and TFIID at genes regulated by both complexes have not been examined. Here we delineated the role of SAGA in the regulation of RNR3 and contrast it to that of TFIID. We find that SAGA components fulfill distinct functions in the regulation of RNR3. The core promoter of RNR3 is SAGA-dependent, and we provide evidence that SAGA, not TAF(II)s within TFIID, are largely responsible for TBP recruitment. This taken together with our previous work provides evidence that SAGA recruits TBP, whereas TFIID mediates chromatin remodeling. Thus, we described an unexpected shift in the division of labor between these two complexes and provide the first characterization of a gene that requires both SAGA and TFIID.
Highlights
Delivery of the TATA-binding protein (TBP) to the core promoter
As described above, SAGA is modular in nature, and its subunits carry out distinct functions (10 –12, 37, 38) such as the covalent modification of histones and TBP recruitment
Because ribonucleotide reductase 3 (RNR3) has a consensus TATA-box, a feature of SAGA-regulated genes [16], we explored the role of SAGA in recruiting TBP to the promoter
Summary
PH499 but taf1–2::TRP1 PH499 but spt3⌬::KanMx PH499 but gcn5⌬::KanMx PH499 but spt7⌬::KanMx PH499 but spt3⌬::KanMx; ⌬gcn5::HIS3 PH499 but (34 bp dA:dT at Ϫ90/Ϫ60) RNR3:LEU2 YJR374 but (34 bp dA:dT at Ϫ90/Ϫ60) RNR3:LEU2 YJR997 but (34 bp dA:dT at Ϫ90/Ϫ60) RNR3:LEU2 YJR1002 but (34 bp dA:dT at Ϫ90/Ϫ60) RNR3:LEU2 YJR1007 but (34 bp dA:dT at Ϫ90/Ϫ60) RNR3:LEU2 the promoter [26]. RNR3 has a consensus TATA-box, and its core promoter is insensitive to TAFII mutations; the TAFII dependence of this gene is mediated by the upstream repression/activating sequences (URS/UAS) [24]. We found that RNR3 is more sensitive to inactivation of the TBP recruitment module than the histone modification module, suggesting that SAGA delivers TBP to the promoter. Consistent with this model, the core promoter of RNR3 is very sensitive to the inactivation of the TBP module of SAGA. We propose a model where the primary function of SAGA is to deliver TBP to the RNR3 core promoter and that of TFIID is to mediate the chromatin remodeling step through the UAS/URS of the gene. We have uncovered unexpected roles of these two coactivator complexes at RNR3, which likely applies to other genes in yeast and metazoans
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