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Distinct Classes of Yeast Promoters Revealed by Differential TAF Recruitment

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Science  19 May 2000:
Vol. 288, Issue 5469, pp. 1242-1244
DOI: 10.1126/science.288.5469.1242

Abstract

The transcription factor TFIID contains the TATA box binding protein (TBP) and multiple TBP-associated factors (TAFs). Here, the association of TFIID components with promoters that either are dependent on multiple TAFs (TAFdep) or have no apparent TAF requirement (TAFind) is analyzed in yeast. At TAFdep promoters, TAFs are present at levels comparable to that of TBP, whereas at TAFindpromoters, TAFs are present at levels that approximate background. After inactivation of several general transcription factors, including TBP, TAFs are still recruited by activators to TAFdeppromoters. The results reveal two classes of promoters: at TAFind promoters, TBP is recruited in the apparent absence of TAFs, whereas at TAFdep promoters, TAFs are co-recruited with TBP in a manner consistent with direct activator-TAF interactions.

TFIID is a general Pol II transcription factor (GTF) that initiates transcription complex assembly by binding to the TATA box through its TBP subunit. TFIID has also been implicated in the mechanism of action of certain promoter-specific activator proteins (activators) (1). Whereas TBP is a general factor, TAFs are highly promoter selective, which raises the question of whether TAFs are co-recruited with TBP to all promoters.

To address this issue we have used TAF temperature-sensitive (ts) mutants (2–7) to identify yeast genes that depend on multiple TAFs or do not require TAFs in general. Results for four representative yeast TAFs—yTAFII145 (2), yTAFII60, yTAFII61/68 (7), and yTAFII17 (4)—are shown in Fig. 1. After ts inactivation of these TAFs, transcription of the RPS5, RPS30,SSB1, ACT1, and RPL25 genes rapidly ceased, whereas transcription of the ADH1,SED1, PGK1, GAL1 (+Gal), andCUP1 (+Cu2+) genes was unaffected (Fig. 1) (8). We refer to these as TAF-dependent (TAFdep) and TAF-independent (TAFind) promoters.

Figure 1

Two classes of promoters defined by their transcriptional requirement for TAFs. Yeast strains harboring ts mutations in one of the TAFs (2, 4, 7) or the Pol II large subunit (22) and their corresponding isogenic wild-type strains were grown at 23°C in 1% yeast extract containing 2% peptone plus glucose or galactose as indicated. After shift to the nonpermissive temperature (37°C) for 1 hour, cells were harvested, and total cellular RNA was prepared. Transcripts were quantitated by primer extension using primers near the transcription start site and the products were separated by denaturing polyacrylamide gel electrophoresis. The CUP1 gene was induced for 15 min by addition of CuSO4 (final concentration, 1 mM) 45 min after the temperature shift.

To determine whether TAFs are differentially recruited to the two classes of promoters, we performed formaldehyde DNA-crosslinking and immunoprecipitation experiments (9). Either α-TAF polyclonal antibodies (Fig. 2A) or an α-influenza hemagglutinin (HA) mouse monoclonal antibody and yeast strains expressing an NH2-terminal triple HA-tagged TAF (10) (Fig. 2B) were used. As expected, TBP bound to all promoters at levels correlating with transcriptional activity (11, 12). However, TAF binding differed according to promoter class. For TAFdep promoters, each TAF bound at levels comparable to that of TBP (Fig. 2A, compare lane 2 with lanes 3 to 6). In contrast, at TAFind promoters, each TAF bound at levels only slightly above background and much lower than that of TBP (Fig. 2A, compare lane 8 with lanes 9 to 12) (13). Other GTFs, such as Pol II, bound to both classes of promoters at comparable levels (Fig. 2C). These results indicate that TAFs are differentially recruited to the two classes of promoters in a manner that correlates with their transcriptional requirement. This general conclusion has been independently reached in a related study (14).

Figure 2

Differential association of TAFs with TAFdep and TAFind promoters. Formaldehyde DNA-crosslinking and immunoprecipitation (IP) analysis (9) was carried out in the wild-type yeast strain W303a (A and C) or in strains expressing a TAF with an NH2-terminal triple influenza HA epitope tag (10). (B) Immunoprecipitation was done with polyclonal antibodies against TBP or a TAF, the mouse monoclonal antibody 16B12 (Covance, Princeton, New Jersey) against HA, or the mouse monoclonal antibody 8WG16 (Covance) against the COOH-terminal domain of Pol II large subunit. Primer pairs located in the core promoter region of each gene were used in the PCR analysis of the immunoprecipitated DNA samples. A PCR fragment corresponding to the internal transcribed region of the GAL4 gene was used as a control for background binding. IP = immunoprecipitation; ORF = open reading frame.

We next investigated the role of TBP and other GTFs in TAF recruitment. Inactivation of TBP by a ts mutant (15) caused a substantial decrease in TBP binding to the ADH1, ACT1, andRPS5 promoters (Fig. 3A, compare lanes 2 and 8); a decrease in Pol II binding (Fig. 3B, compare lanes 3 and 4); and a decrease in transcription. Remarkably, however, inactivation of TBP did not abolish binding of TAFs to the TAFdep promoters of the RPS5 andACT1 genes (Fig. 3A, compare lanes 3 to 6 with lanes 9 to 12) (16).

Figure 3

Requirement of TBP and activators for recruitment of TAFs to TAFdep promoters. (A) Requirement of TBP for recruitment of TAFs. The TBP ts mutant (15) and isogenic wild-type yeast strains were grown at 23°C and shifted to 37°C for 1 hour before formaldehyde crosslinking. (B) Requirement of TBP for recruitment of Pol II. (C) Requirement of activators for recruitment of TBP and TAFs. Promoter derivatives containing the intact RPS5 promoter (−592 to +59) or the core promoter sequence (−135 to +59) fused to aLacZ sequence were integrated at the URA3 locus. The resulting yeast strains were used in formaldehyde crosslinking and immunoprecipitation analysis. The primers used for the PCRs are indicated.

The unexpected finding that recruitment of TAFs to TAFdep promoters does not require TBP raised the possibility that TAFs might be constitutively bound to the core promoter, an element that in some cases confers TAF dependence (17). To address this possibility, we asked whether promoter-specific transcriptional activators were required for association of TAFs with the RPS5 promoter. Association of TAFs and TBP was lost after removal of the activator binding sites from the RPS5 promoter (Fig. 3C), which indicates that activators are required for TAF recruitment.

Finally, we analyzed the role of representative GTFs in recruitment of TBP and TAFs. Inactivation of TFIIB (11) or suppressor of RNA polymerase B-4 (Srb4) (18) dramatically reduced binding of TBP to the TAFind ADH1 promoter, as previously reported (11), but had little effect on binding of TBP to the TAFdep promoters of the ACT1and RPS5 genes (Fig. 4).

Figure 4

Requirement of TFIIB and Srb4 for recruitment of TBP and TAFs to TAFdep promoters. TFIIB (11) and Srb4 (18) ts mutant strains were grown at 23°C and shifted to 37°C for 1 hour; association of TBP and TAFs was analyzed as in Fig. 3.

In summary, we have identified two distinct classes of yeast promoters based on their requirement for TAFs. At TAFdeppromoters, TAFs are recruited and are required for delivery of TBP (19). Recruitment of TAFs to TAFdeppromoters is activator dependent but appears to be relatively independent of other GTFs—surprisingly, even TBP. These data are consistent with the possibility that, at TAFdeppromoters, TAFs are directly targeted by activators, which results in recruitment of TAFs and TBP. The notion that TAFs may be direct targets of some activators is consistent with a variety of biochemical studies (1).

For TAFind promoters, TAFs are not required for transcriptional activity or for TBP recruitment (19). The same assay that revealed the approximate stoichiometric association of TAFs and TBP with TAFdep promoters showed that the level of TAF association with these promoters is close to background. These results strongly suggest that TBP is recruited to TAFind promoters in the absence of TAFs, perhaps alone or in a complex with other proteins (20). The absence of TAFs on TAFind promoters in wild-type cells confirms their dispensability for transcription of certain genes, a conclusion independently derived from yeast TAF inactivation studies (2–6, 21).

Although recruitment of TBP to TAFind promoters does not require TAFs, there is a strong dependence on GTFs, such as TFIIB and Srb4. These results are consistent with the possibility that, at TAFind promoters, the activator targets one of these GTFs, either directly or through another component, which through cooperative interactions ultimately promotes TBP binding and transcription.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: michael.green{at}umassmed.edu

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