The yeast GAL11 protein binds to the transcription factor IIE through GAL11 regions essential for its in vivo function.

Abstract

The GALlJ gene encodes an auxiliary transcription factor required for full expression of many genes in yeast. The GALI-encoded protein (Galllp) has recently been shown to copurify with the holoenzyme of RNA polymerase II. Here we report that Galllp stimulates basal transcription in a reconstituted transcription system composed of recombinant or highly purified transcription factors, TFIIB, TFIIE, TFIIF, TFIIH, and TATA box-binding protein and core RNA polymerase II. We further demonstrate that each of the two domains of Galllp essential for in vivo function respectively participates in the binding to the small and large subunits of TFIIE. The largest subunit of RNA polymerase II was coprecipitated by anti-hemagglutinin epitope antibody from crude extract of GALM wild type yeast expressing hemagglutinintagged small subunit of TFIIE. Such a coprecipitation of the RNA polymerase subunit was seen but in a greatly reduced amount, if extract was prepared from gallI null yeast. In light of these findings, we suggest that Galllp stimulates promoter activity by enhancing an association of TFIIE with the preinitiation complex in the cell.The GALl] gene of the yeast Saccharomyces cerevisiae is required for full expression of many but not all genes. Lossof-function mutations of GALl] result in pleiotropic defects but not in lethality on rich medium (1-4). Biochemical analyses indicated that the protein encoded by GALl] (Galllp) stimulates basal transcription in cell-free systems developed from yeast whole cell or nuclear extracts and suggested a direct interaction between Gall lp and a basal transcription factor(s) (5, 6). As a support of this idea, it has recently been found that Gall lp copurifies with "holoenzyme" of RNA polymerase II (PolIl), which is composed of core PolIl, some of the basal transcription factors, nine SRB (suppressor of RNA polymerase B) proteins (7,8), global transcription factors Sin4p (9) and Rgrlp (10), and over a dozen of as yet unknown proteins (11)(12)(13)(14). The PollI holoenzyme is capable of catalyzing basal as well as activator-dependent transcription from various genes in vitro in the presence of the remaining basal factors, and therefore it is assumed to be a preassembled form of the preinitiation complex in vivo (7,15). Components of the yeast holoenzyme, however, vary depending on the purification strategy to some extent (7,11,12). Nevertheless, TATA box-binding protein (TBP) and TFIIE are always missing, while Galllp is universally present in these enzyme preparations (11-13). By contrast, mammalian Polll holoenzyme, which has recently been reported to exist in rat liver cells (16), contains all components required for promoter-specific transcription initiation.In the present study, we show that Galllp stimulates basal transcription in a highly purified reconstituted transcription system. We also demonstrate that Gall ip makes contacts with TFIIE through Gall lp regions that are essential for its in vivo function. These results lead us to hypothesize that Galllp contributes to...

Core promoter elements are essential as selective determinants for function of the yeast transcription factor GAL11

“…It has been reported that the holoenzyme of RNA polymerase II, in which Gall lp is a component, enhances basal as well as activated transcription in a reconstituted transcription system [3,4,17,18]. Our recent experiments indicated that Gall lp directly interacts with TFIIE, and further suggested that it is through this interaction that Galllp regulates the promoter activity [19]. We may therefore suggest that Gall lp in the holoenzyme regulates transcription depending on the presence of the TATA box through interaction with TFIIE.…”

“…This notion has been supported by the recent finding that Galllp copurifies with the RNA polymerase II holoenzyme [17,18]. In fact, immunoprecipitation experiments with antiGall lp antibody have demonstrated that Gall lp coprecipitates with the largest subunit of RNA polymerase II [19].…”

Drosophila Mediator Complex Is Broadly Utilized by Diverse Gene-Specific Transcription Factors at Different Types of Core Promoters

“…Related with this idea, it has been reported that in yeast, recruitment of general transcription factors such as TBP, TFIIB, and TFIIH to active promoters requires the function of Mediator (17,25,32). Also, TFIIE interacts with the Mediator protein Gal11 (34). Further analyses will be required to clarify whether these interactions, observed both in yeast and Drosophila, participate in the control of the stepwise preinitiation complex assembly in the course of transcription activation or simply reflect the affinities between the components of preassembled Pol II holoenzyme.…”

A target essential for the activity of a nonacidic yeast transcriptional activator

“…Deletion of Gal11 residues 866-929 (lines 9 and 10) had an effect equivalent to total deletion of Gal11 (lines 3 and 4). This part of Gal11 (i.e., residues 866-929) is required for whatever general role it plays in the mediator (31). These results, taken together, suggest that LexAϩP201 contacts Gal11 somewhere in the segment encompassed by residues 187-618.…”