In yeast, the TBP-associated factors (TAFs) Taf17, Taf60, and Taf61(68) resemble histones H3, H4, and H2B, respectively. To analyze their roles in vivo, conditional alleles were isolated by mutagenizing their histone homology domains. Conditional alleles of TAF17 or TAF60 can be specifically suppressed by overexpression of any of the other histone-like TAFs. This and other genetic evidence supports the model of a histone octamer-like structure within TFIID. Shifting strains carrying the conditional TAF alleles to non-permissive conditions results in degradation of TFIID components and the rapid loss of mRNA production. Therefore, in contrast to previous studies in yeast that found only limited roles for TAFs in transcription, we find that the histone-like TAFs are generally required for in vivo transcription.
Many questions remain concerning the role of TFIID TBP-associated factors (TAFs) in transcription, including whether TAFs are required at most or only a small subset of promoters. It was shown previously that three histone-like TAFs are broadly required for transcription, but interpretation of this observation is complicated because these proteins are components of both TFIID and the SAGA histone acetyltransferase complex. Here we show that mutations in the yeast TFIID-specific protein Taf40 lead to a general cessation of transcription, even in the presence of excess TBP, suggesting that the TFIID complex is required at most promoters in vivo. Received May 6, 1999; revised version accepted August 11, 1999. RNA polymerase II (Pol II) is positioned on a promoter by a set of accessory basal transcription factors. One of these factors, TFIID, binds the consensus promoter sequence TATAA and nucleates the assembly of other transcription factors. One subunit of TFIID, the TATAbinding protein (TBP), is necessary and sufficient for this activity in vitro. However, TBP in vivo is associated with several multisubunit complexes that mediate transcription by RNA polymerase I, II, or III (Hernandez 1993). The TBP-containing complexes implicated in Pol II transcription are TFIID, the TBP-MOT1 complex (Auble and Hahn 1993), and the SNAPc complex in higher eukaryotes (Hernandez 1993). In addition, the SAGA histone acetyltransferase complex can associate with TBP transiently (Eisenmann et al. 1992;Sterner et al. 1999).The TFIID complex consists of TBP and 10-12 TBPassociated factors (TAFs). The essential role of TBP in Pol II-mediated transcription has been demonstrated both in vivo and in vitro (Hampsey 1998). The functions of TAFs in transcription are less clear (for review, see Verrijzer and Tjian 1996;Hoffmann et al. 1997;Hahn 1998). In vitro experiments in mammalian, Drosophila, or yeast systems have indicated a requirement for TAFs in responding to transcriptional activators but not for basal transcription (Kokubo et al. 1993;Chen et al. 1994;Reese et al. 1994). However, activated transcription in vitro has also been reported in a yeast system apparently lacking TAFs, with activation being mediated by the Pol II-associated mediator/SRB proteins (Kim et al. 1994;Koleske and Young 1994). TAF-independent activation has also been reported in mammalian in vitro transcription systems (Oelgeschlager et al. 1998;Wu et al. 1998). Therefore, there may be both TAF-dependent and -independent activation mechanisms that can be emphasized by the particular choice of in vitro transcription system. In vivo experiments are therefore essential for testing the physiological relevance of in vitro results.Several other roles for TAFs have been documented in addition to their proposed function as transcriptional coactivators. TFIID makes extensive contacts with core promoter elements in addition to the TATA element, and these contacts are made by the TAFs (for review, see Hoffmann et al. 1997). Furthermore, the largest TAF can inhibit TBP bin...
We have shown previously that a TFII-I-related protein, hMusTRD1/BEN, represses transcriptional activity of TFII-I. The repression by hMusTRD1/BEN was hypothesized to occur via a two-step competition mechanism involving a cytoplasmic shuttling factor and a nuclear cofactor required for transcriptional activation of TFII-I. Employing a two-hybrid approach with both yeast genomic and mouse cDNA libraries in parallel, we have identified the RING-like zinc finger containing Miz1/PIASx/Siz2, which is a ubiquitin-protein isopeptide ligase in the SUMO pathway, as the potential nuclear cofactor that interacts with both TFII-I and hMus-TRD1/BEN. Our conclusion is based on the following observations. First, the interactions are biochemically confirmed in mammalian cells where Miz1/mPIASx interacts with both TFII-I and hMusTRD1/BEN when these proteins are ectopically co-expressed. Second, coexpression of a nuclear localization signal-deficient mutant of Miz1/mPIASx with wild type TFII-I fails to alter the subcellular localization of the former. Finally, ectopically expressed Miz1/mPIASx augments the transcriptional activity of TFII-I and relieves the repression exerted by a mutant hMusTRD1/BEN that co-localized with TFII-I in the nucleus.TFII-I belongs to a family of proteins characterized by the presence of I-repeats first identified in the founding member TFII-I (1-8). Although TFII-I functions as a signal-induced transcriptional activator, the role of the related family member hMusTRD1/BEN is less clear. Both TFII-I and hMusTRD1/ BEN are mapped to the breakpoint regions of the 7q11.23 Williams-Beuren syndrome deletion (reviewed in Ref. 2). Furthermore, genetic and biochemical analyses show that each of these proteins have multiple isoforms both in mice and in humans (7, 9 -11). The transcription functions of hMusTRD1/ BEN have not yet been well characterized biochemically. It was first reported (3) as a muscle specific activator of the troponin I gene. It also seems to function as an activator in yeast onehybrid assays (6). However, clear demonstration of its activator function is not yet obtained. Results from our laboratory suggest that hMusTRD1/BEN behaves as a specific repressor of TFII-I (10). Although each protein when individually expressed exhibits predominant nuclear localization in eukaryotic cells, TFII-I is excluded from the nucleus when it is co-expressed with hMusTRD1/BEN. Nuclear exclusion of TFII-I results in the repression of the TFII-I-responsive c-fos gene. A key to this novel nuclear exclusion function appears to be the serine stretch (ss) 1 in hMusTRD1/BEN because deletion of this stretch results in co-occupancy of both proteins in the nucleus (10). However, although the ⌬ss hMusTRD1/BEN failed to prevent nuclear localization of TFII-I, the transcriptional repression of TFII-I was still observed. The latter data led us to postulate that the repression by hMusTRD1/BEN involves the following two-step mechanism: a competition for a common cytoplasmic factor required for nuclear translocation and a ...
In S. cerevisiae, K + transport relies principally on two structurally related membrane proteins, known as Trk1p and Trk2p. Direct involvement in cation movements has been demonstrated for Trk1p, which is a high-affinity K + transporter. Initially described as a low-affinity K + transporter, Trk2p seems to play a minor role in K + transport, since its activity is only apparent under very specific conditions, such as in a ∆sin3 background. Here we show that growth of a ∆trk1∆sin3 double mutant, under K + -limiting conditions or at low pH, is Trk2p-dependent, and by Northern blot analysis we demonstrate that deletion of SIN3 results in transcriptional derepression of TRK2. In addition, we show that heterologous overexpression of TRK2 with the inducible GAL1 promoter bypasses Sin3p repression in a ∆trk1∆trk2 double mutant and fully restores growth under non-permissive conditions. Furthermore, kinetic experiments in a ∆trk1∆sin3 double mutant revealed a K + transporter with an apparent high affinity and a moderate capacity. Taken together, these results indicate that TRK2 encodes a functional K + transporter that, under our experimental conditions, displays distinctive kinetic characteristics.
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