RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene.

Abstract

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAPlbinding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAPl-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1-and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.Transcription of the Saccharomyces cerevisiae HIS4 gene is regulated by two separate systems. One system, general amino acid control, increases HIS4 transcription upon starvation for any amino acid(s) (17). In this system, amino acid starvation causes an increase in the levels of the GCN4 protein (for a review, see reference 13). GCN4 stimulates transcription by binding directly to the promoters of genes that are under general control (1, 15). In vitro, GCN4 binds to five 5'-TGACTC-3' sites located within the HIS4 promoter but has a much higher affinity for one of these sites, site C, than for the other four sites (1). The GCN4 protein is also expressed at low levels under nonstarvation conditions and is responsible for some of the H1S4 transcription under these conditions.The second system that regulates HIS4 transcription is basal control. Basal control activates HIS4 transcription in the absence of amino acid starvation but further stimulates HIS4 transcription under conditions of phosphate or adenine limitation (2, 28). Basal-control regulation of HIS4 requires two trans-acting proteins, BAS1 and BAS2 (PH02). BAS1 has a DNA-binding region that is similar to Myb proteins of higher organisms (28), and BAS2 contains a region that is similar to the homeo box (7). Neither BAS1 nor BAS2 alone can activate HIS4 transcription. BAS1 and BAS2 bind adjacently on the HIS4 promoter at a site upstream of the high-affinity GCN4 site C (28). BAS1 and BAS2 can activate HlS4 transcription independently of GCN4. Likewise, GCN4 can activate HIS4 transcription independently of both BAS1 and BAS2.A yeast strain that does not contain GCN4, BAS1, and BAS2 (gcn4-2 basl-2 bas2-2) is His-as a result of lack of transcription of the HIS4 gene (3). Therefore, normal activation of HIS4 transcrip...

Alternative Mechanisms of Transcriptional Activation by Rap1p

“…In contrast, transcription of constructs with two adjacent sites was reduced, rather than increased, by histone depletion. The synergistic activation by adjacent Rap1p-binding DNA may thus be the relief of the transcriptional repression by histones, as suggested elsewhere (59,60). The 40% decrease of transcription observed for plasmids with two Rap1p DNA-binding sites in histone-depleted conditions may indicate a specific enhancement of transcriptional response mediated by the presence of histones (61).…”

Artificially Recruited TATA-Binding Protein Fails To Remodel Chromatin and Does Not Activate Three Promoters That Require Chromatin Remodeling

“…These data therefore suggest that two functions, chromatin opening and recruitment of the general transcriptional machinery, are required to achieve transcriptional activation. Similarly, RAP1 is required for transcription of HIS4 independent of BAS1, BAS2, and GCN4 in sit mutants, and sit1 and sit2 encode mutant alleles of the two largest subunits of RNA polymerase II (15). Thus, chromatin opening by RAP1 is likely also necessary for activator-independent transcription of HIS4 by these mutant alleles of RNA polymerase II.…”

“…Activation of this promoter by GCN4 requires a RAP1 binding site (15), although RAP1 cannot activate HIS4 by itself; rather, RAP1 appears to function at HIS4 to open the chromatin to allow access by GCN4 (15,82). Chromatin opening by RAP1 is also needed for HIS4 activation by GAL4 at a weak binding site, but not at a strong binding site (82).…”

The histone methylase Set2p and the histone deacetylase Rpd3p repress meiotic recombination at the HIS4 meiotic recombination hotspot in Saccharomyces cerevisiae

“…One role of these transcription factors is presumably to recruit chromatin-modifying complexes to the HIS4 promoter. The transcription factor Rap1p directly alters the positioning of nucleosomes in the HIS4 upstream region (21,22). Based on the evidence that HIS4 hotspot activity is likely related to a particular chromatin structure, in this study, we analyzed the effects of mutations affecting chromatin modifications on HIS4 hotspot activity.…”