A complex regulatory element from the yeast gene ENO2 modulates GCR1-dependent transcriptional activation.

Abstract

The GCR1 gene product is required for maximal transcription of many yeast genes including genes encoding glycolytic enzymes. Transcription of the yeast enolase gene EN02 is reduced 50-fold in strains carrying a gcrl null mutation. cis-acting sequences that are sufficient for GCRI-dependent regulation ofEN02 expression were identified by using an enhancerless CYC1 promoter which is not normally dependent on GCR1 for expression. A 60-bp EN02 sequence that was sufficient to provide high-level, GCRI-dependent transcriptional activation of the CYCi promoter was identified. This 60-bp element could be subdivided into a 30-bp sequence containing a novel RAPl-binding site and a GCR1-binding site which did not activate CYCi transcription and a 30-bp sequence containing a novel enhancer element that conferred moderate levels of GCRI-independent transcriptional activation. (12,24,30,36,39,45,53,55,58). RAPl-binding sites were mapped within UAS elements of the glycolytic genes PGK1 (13), PYKI (43), PDC1 (11), TPI1 (49), EN01 (7), EN02 (7); also see below), and TDH3 (5 mRNA are reduced 20-to 50-fold in strains carrying a gcrl null mutation, suggesting that GCR1 modulates transcription of these genes. Several genes whose expression depends on the GCR1 gene product contain a nucleotide sequence motif (CTICC), often located adjacent to a RAPl-binding site (5, 10,11,13,48,49). It was shown for the EN01, PYK1, PGK1, and TDH3 genes that the CTTCC sequence is required for maximal RAPI-dependent UAS element activity (5, 10, 13) (the CTTCC sequence is also required for full RAPl-dependent activation of the RNR2 gene; however, the GCR1 dependence of this gene remains to be determined [36]). The GCR1 protein has recently been shown to bind specifically to C1TTlCC and CATCC sequences (4, 35) within the 5' regulatory region of several yeast glycolytic genes, suggesting that the GCR1 protein may function with RAP1 to activate glycolytic gene expression.Transcription of the yeast enolase genes EN01 and EN02 is regulated by multiple UAS elements (7,17,18). We showed previously that deletions within the regulatory regions of the EN01 and EN02 genes rendered expression of these genes independent of GCRI (reference 31 and unpublished results). This suggests that GCR1-dependent activation of these genes is not direct but, rather, that the GCR1 gene product must function to overcome an inhibitor of transcription. This also suggests that regulation by GCR1 involves at least two cis-acting elements, one positive and one negative.Here we report the identification and characterization of EN02 sequences that are required for GCR1-dependent activation of transcription. We show that GCR1-dependent activation of transcription involves interactions among multiple cis-acting sites and trans-acting factors. The role of the GCR1 gene product in the activation mechanism is discussed. MATERIALS AND METHODSStrains, growth conditions, and yeast transformation. Saccharomyces cerevisiae S173-6B (a leu2-3 leu2-112 his3-1 trpl-289 ura3-52) (41) was used as the wil...

Activation of gastrin gene transcription in islet cells by a RAP1‐like cis‐acting promoter element

“…The RAP1 binding site in the gastrin promoter is adjacent to cis-regulatory elements which bind transcriptional repressors (ATTCCTCT) or activators (CATATGG). This is consistent with previous studies in yeast suggesting that the regulatory function of RAP1 is determined by the context of its binding site and, presumably its interaction with other factors [36,37]. Gel mobility shift assays and methylation interference studies also demonstrated sequence specific DNA-protein interactions between the gastrin DNA element and recombinant RAP1 protein identical to those reported for RAP1 binding sites in yeast promoters.…”

Mutations in an Abf1p binding site in the promoter of yeast RPO26 shift the transcription start sites and reduce the level of RPO26 mRNA

“…For quantitation of RP026 mRNA, the amount of signal generated from both mutant and wild-type initiation sites (indicated by brackets) were quantitated for lanes 2 to 4. for the ADHl gene amongst different wild-type yeast strains (Bennetzen and Hall, 1982;Pinto et al, 1992Pinto et al, , 1994. The role of Abflp binding-sites in the expression of a variety of genes has been studied (Buchman and Kornberg, 1990;Della Seta et al, 1990a,b;Dequard-Chablat et al, 1991;Halfter et al, 1989;Hamil et al, 1988;Herruer et al, 1989;Kraakman et al, 1991;Mager and Planta, 1990;Sinclair et al, 1994;Trawick et al, 1992;Willett et al, 1993); however, to our knowledge, this is the first report of a shift in the initiation sites of transcription due to a mutation in an Abflp binding-site. In other studies, promoters have often been used to drive expression of the lacZ reporter gene and the effects of Abfl p binding-site mutations have been analysed by monitoring b-galactosidase activity in cellular extracts.…”

“…For these genes Abflp appears to facilitate the access of other regulatory factors to promoters, thereby mediating their regulatory effects (GonCalves et al, 1995;Trawick et al, 1992;Willett et al, 1993). The ability of Abflp to create a sharp bend in DNA (McBroom and Sadowski, 1994) and to position nucleosomes on the promoter (De Winde et al, 1993) might underlie the mechanism by which it facilitates access to DNA for other regulatory factors.…”

“…Abflp alone is not a potent transcriptional activator, normally requiring the cooperation of other weak activators (Buchman and Kornberg, 1990;Della Seta et a/., 1990b;Halfter et al, 1989;Hamil eta/., 1988;Kraakman et a/., 1991;Sinclair et al, 1994;Trawick et al, 1992;Willett et al, 1993). For example, Abflp binding-sites are often associated with a T-rich sequence, which functions in a variety of yeast promoters as a transcriptional activator (Buchman and Kornberg, 1990;GonCalves et al, 1995;Iyer and Struhl, 1995;Lue et al, 1989;Schultes and Szostak, 1991).…”

“…Abfl p is a highly abundant transcriptional activator and is required for constitutive o r regulated transcription of a large number of genes (Buchman and Kornberg, 1990;Della Seta et al, 1990b;Dhawale and Lane, 1993;GonCalves et al, 1995;Halfter et al, 1989;Hamil et al, 1988;Herruer et al, 1989;Kraakman et al, 1991;Mager and Planta, 1990;Sinclair et al, 1994;Trawick et al, 1992;Willett et al, 1993). For these genes Abflp appears to facilitate the access of other regulatory factors to promoters, thereby mediating their regulatory effects (GonCalves et al, 1995;Trawick et al, 1992;Willett et al, 1993).…”