Impaired Core Promoter Recognition Caused by Novel Yeast TAF145 Mutations Can Be Restored by Creating a Canonical TATA Element within the Promoter Region of the TUB2Gene

Abstract

The general transcription factor TFIID, which is composed of TATA-binding protein (TBP) and an array of TBP-associated factors (TAFs), has been shown to play a crucial role in recognition of the core promoters of eukaryotic genes. We isolated Saccharomyces cerevisiae yeast TAF145 (yTAF145) temperature-sensitive mutants in which transcription of a specific subset of genes was impaired at restrictive temperatures. The set of genes affected in these mutants overlapped with but was not identical to the set of genes affected by a previously reported yTAF145 mutant (W.-C. Shen and M. R. Green, Cell 90:615-624, 1997). To identify sequences which rendered transcription yTAF145 dependent, we conducted deletion analysis of the TUB2 promoter using a novel mini-CLN2 hybrid gene reporter system. The results showed that the yTAF145 mutations we isolated impaired core promoter recognition but did not affect activation by any of the transcriptional activators we tested. These observations are consistent with the reported yTAF145 dependence of the CLN2 core promoter in the mutant isolated by Shen and Green, although the CLN2 core promoter functioned normally in the mutants we report here. These results suggest that different promoters require different yTAF145 functions for efficient transcription. Interestingly, insertion of a canonical TATA element into the TATA-less TUB2 promoter rescued impaired transcription in the yTAF145 mutants we studied. It therefore appears that strong binding of TBP to the core promoter can alleviate the requirement for at least one yTAF145 function.In eukaryotes, transcriptional initiation by RNA polymerase II requires a set of general transcriptional factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) (reviewed in references 16, 72, and 78) and the SRB-MED complex associated with the carboxy-terminal domain of RNA polymerase II (reviewed in references 65 and 66). These factors nucleate on the core promoter of eukaryotic class II genes to form a preinitiation complex in an ordered stepwise fashion (reviewed in references 9, 23, and 78) or are recruited in a simpler sequence involving a small number of preassembled units (reviewed in references 43 and 73). In either case, the first step, which is the sequence-specific binding of TFIID (76), is thought to be a major rate-limiting step during transcription and a focal point for the activity of transcriptional activators (14,40,54).TFIID is a multiprotein complex composed of TATA-binding protein (TBP) and an array of TBP-associated factors (TAFs); in total, the complex includes 8 to 12 molecules ranging in size from 15 to 250 kDa (reviewed in references 11, 51, 91, and 93). Almost all of these TAFs are conserved among evolutionarily divergent organisms (humans, Drosophila melanogaster, and Saccharomyces cerevisiae), albeit with a few exceptions (for instance, no orthologue of Drosophila TAF110 or human TAF130 (dTAF110/hTAF130) is found in yeast). This level of conservation suggests that TAFs play a fundamental role in eukaryotic transcription (review...

Requirement for Yeast TAF145 Function in Transcriptional Activation of the RPS5 Promoter That Depends on Both Core Promoter Structure and Upstream Activating Sequences

“…11). Promoter swapping experiments demonstrated that TAF145 function is demanded by the core promoter region rather than by upstream activating sequences (UASs) as examined for the CLN2, RPS5, and TUB2 genes (19,20). Thus, it appears that TAF145 function is tightly connected to core promoter recognition, in accordance with human TAF250, which directly recognizes an initiator element as described above (13).…”

“…The yeast strains used in this study, YTK3010 (wild type), YTK3002 (Y570N), YTK3003 (N568⌬), and YTK3005 (T657K) were generated by plasmid shuffle techniques from the parental strain Y22.1 (20). They carry a deletion of the chromosomal TAF145 coding region and the wild type or mutant TAF145 gene on a TRP1-based low copy number vector (20).…”

“…1 and UAS GAL ϩCYC1/-174 in Fig. 5) were described previously (20). pM1452 and pM1591 were subjected to site-specific mutagenesis (43) to create pM3226 (shown as ⌬CLN2 GAGA in Fig.…”

“…We recently isolated two novel temperature-sensitive taf145 mutants in which the expression profiles of some genes were not identical to those in previously reported taf145 mutants (20). In our mutants, the core promoter of theTUB2 gene failed to mediate basal transcription, but the function was restored by inserting a consensus TATA element (20).…”

A role of the TATA box and the general co-activator hTAFII130/135 in promoter-specific trans-activation by simian virus 40 small t antigen

“…TFIID comprises the TATA box-binding protein (TBP) and about 12 TBP-associated factors (TAF II s). Although referred to as general transcription factors, distinct TAF II proteins seem to be recruited selectively by specific promoters and are, therefore, essential for the transcription of a subset of genes (Albright & Tjian, 2000;Tsukihashi et al, 2000;Li et al, 2002;and references therein). In addition to these general transcription factors, sequence-specific DNA-binding transcription factors, co-activators and chromatin-remodelling proteins are required to ac2tivate transcription (reviewed by Näär et al, 2001).…”