The function of Spt3, a subunit of the SAGA complex, in PGK1 transcription is restored only partially when reintroduced by plasmid into taf1 spt3 double mutant yeast strains

In Saccharomyces cerevisiae, class II gene promoters contain two classes of TATA elements: the TATA box and the TATA-like element. Functional loss of TFIID and SAGA transcription complexes selectively impacts steady-state mRNA levels expressed from TATA-like element-containing (i.e., TATA-less) and TATA box-containing promoters, respectively. While nascent RNA analysis has revealed that TFIID and SAGA are required for both types of promoters, the division of their roles remains unclear. We show here that transcription from the PGK1 promoter decreased in some cases by more than half after disruption of the TATA box or SPT3 (spt3Δ), whereas spt3Δ did not affect transcription from the TATA-less promoter, consistent with the prevailing view that Spt3 functions specifically in a TATA box-dependent manner. Transcription from this promoter was abolished in the spt3Δ taf1-N568Δ strain but unaffected in the taf1-N568Δ strain, regardless of TATA box presence, suggesting that TFIID was functionally dispensable for PGK1 transcription at least in the SPT3 strain. Furthermore, transcription from the TATA box-containing PGK1 promoter was slightly reduced in the taf1 strain lacking TAND (taf1-ΔTAND) upon temperature shift from 25 to 37 ℃, with restoration to normal levels within 2 h, in an Spt3-dependent manner. Interestingly, when SPT3 was reintroduced into the spt3Δ TAF1, spt3Δ taf1-N568Δ or spt3Δ taf1-ΔTAND strain, TATA box-dependent transcription from this promoter was largely restored, but TFIID independence in transcription was not restored, especially from the TATA-less promoter, and transient TAND/Spt3-dependent fluctuations of transcription after the temperature shift were also not recapitulated. Collectively, these observations suggest that Spt3 has multiple functions in PGK1 transcription, some of which may be intimately connected to Taf1 function and may therefore become unrestorable when the TFIID and SAGA functions are simultaneously compromised.