Positive and negative functions of the SAGA complex mediated through interaction of Spt8 with TBP and the N-terminal domain of TFIIA

Abstract

A surface that is required for rapid formation of preinitiation complexes (PICs) was identified on the N-terminal domain (NTD) of the RNA Pol II general transcription factor TFIIA. Site-specific photocross-linkers and tethered protein cleavage reagents positioned on the NTD of TFIIA and assembled in PICs identified the SAGA subunit Spt8 and the TFIID subunit Taf4 as located near this surface. In agreement with these findings, mutations in Spt8 and the TFIIA NTD interact genetically. Using purified proteins, it was found that TFIIA and Spt8 do not stably bind to each other, but rather both compete for binding to TBP. Consistent with this competition, Spt8 inhibits the binding of SAGA to PICs in the absence of activator. In the presence of activator, Spt8 enhances transcription in vitro, and the positive function of the TFIIA NTD is largely mediated through Spt8. Our results suggest a mechanism for the previously observed positive and negative effects of Spt8 on transcription observed in vivo.[Keywords: Transcription; SAGA; TFIIA; coactivator; repression] Supplemental material is available at http://www.genesdev.org.

The S. pombe SAGA complex controls the switch from proliferation to sexual differentiation through the opposing roles of its subunits Gcn5 and Spt8

“…Rather, based on our finding that the spt8⌬ activation defect is suppressed by gcn5⌬, Spt8 appears to inhibit the repressive activity of Gcn5. Previous studies in S. cerevisiae have implicated Spt8 in transcriptional activation by recruitment of TBP (Eisenmann et al 1994;Belotserkovskaya et al 2000;Bhaumik and Green 2002;Warfield et al 2004;Sermwittayawong and Tan 2006;Laprade et al 2007). It is not clear how such an activity fits with our results and raises the possibility of other mechanisms by which Spt8 can control transcription.…”

“…Second, Spt3 and Spt8 recruit the general factor TATA-binding protein (TBP) to certain promoters (Dudley et al 1999;Green 2001, 2002;Larschan and Winston 2001). Spt3 and Spt8 also have been shown to negatively regulate the recruitment of TBP (Belotserkovskaya et al 2000;Yu et al 2003;Warfield et al 2004). Third, Ubp8 is a histone deubiquitylase for histone H2B at K123 (Henry et al 2003; K.K.…”

Transcriptional Regulation inSaccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators

“…Gene expression studies have also shown that Gcn5 and subunits of the SAGA TBP-binding module have opposing roles; S. cerevisiae SPT3 and GCN5 mutations were found to have opposite effects in transcriptional regulation of the HO and STE11 genes (Yu et al 2003;Helmlinger et al 2008), although it is possible that some of these phenotypes are due to indirect effects. SAGA can also repress basal expression of some genes in vitro and in vivo (Belotserkovskaya et al 2000;Warfield et al 2004).…”

The Transcriptional Repressor Activator Protein Rap1p Is a Direct Regulator of TATA-binding Protein

“…These include NC2, TFIIA, TFIIB (77), Mbf1p (78), Spt3p, Spt8p, and Ada1p subunits of SAGA (79,80), TFIID Taf1p and TAND (58,59,81,82), Med8p and Med20p subunits of Mediator (83), Brf1p subunit of TFIIIB (84), TAF I 48 subunit of SL1 (85), N-terminal domain of Mot1p (86), SNAP190 subunit of SNAP C (87), and the NOT complex (88). Interestingly, many of these TBP-targeted factors both activate and repress transcription (76).…”