Upstream activating sequence

About: Upstream activating sequence is a research topic. Over the lifetime, 1633 publications have been published within this topic receiving 100112 citations.

Promoter sequences regulated by the calcineurin-activated transcription factor Crz1 in the yeast ENA1 gene.

Abstract: In Saccharomyces cerevisiae the transcription of the ENA1 gene is modulated by multiple transduction pathways that respond to osmotic, ionic and nutrient stresses. We have investigated the molecular mechanisms involved in ENA1 induction by the calcium-calcineurin-activated transcription factor Crz1/Tcn1. We found in the ENA1 promoter a calcium-responsive, Crz1-dependent upstream activating region (UASENA1) located between –713 bp and –826 bp relative to the translation start. This region contains two separate control elements: the upstream element (5′-GAATGGCTG-3′) between –813 and –821 binds Crz1p with lower affinity and mostly contributes to basal ENA1 expression, whereas the downstream element (5′-GGGTGGCTG-3′) between –727 and –719 binds Crz1p with higher affinity and is a major determinant of the induction response to calcium.

cAMP-dependent phosphorylation and inactivation of yeast transcription factor ADR1 does not affect DNA binding.

Abstract: Transcription factor ADR1 increases the level of ADH2 gene expression 200-fold by binding to a palindromic upstream activation sequence (UAS1) in the glucose-repressible ADH2 promoter in Saccharomyces cerevisiae. cAMP-dependent protein kinase (cAPK) phosphorylates ADR1 in vitro and a yeast strain with elevated cAPK activity inhibits the ability of ADR1 to activate ADH2 transcription in vivo [Cherry, J. R., Johnson, T. R., Dollard, C., Schuster, J. R. & Denis, C. L. (1988) Cell 56, 409-419]. Intact ADR1 protein was detected at comparable levels in extracts made from repressed or derepressed yeast cells, indicating that glucose repression is not due to absence of ADR1. ADR1 in extracts made from glucose-repressed and -derepressed cells bound UAS1 DNA with similar affinities despite having greatly different abilities to activate ADH2 gene expression in vivo. A mutant form of ADR1 encoded by ADR1-5c, which has an altered consensus sequence for phosphorylation by cAPK conferred constitutive expression on ADH2 but bound DNA to the same extent as wild-type ADR1 protein. Similarly, normal DNA binding was seen for ADR1 produced in mutants with altered levels of cAPK activity. Because inactivation of ADR1 by phosphorylation has no detectable effect on either DNA binding or ADR1 levels, ADR1 probably binds to UAS1 constitutively and phosphorylation prevents it from promoting transcription.

Cloning, nucleotide sequence, and regulation of MET14, the gene encoding the APS kinase of Saccharomyces cerevisiae.

Abstract: The MET14 gene of Saccharomyces cerevisiae, encoding APS kinase (ATP: adenylylsulfate-3′-phosphotransferase, EC 2.7.1.25), has been cloned. The nucleotide sequence predicts a protein of 202 amino acids with a molecular mass of 23060 dalton. Translational fusions of MET14 with the β-galactosidase gene (lacZ) of Escherichia coli confirmed the results of primer extension and Northern blot analyses indicating that the ca. 0.7 kb mRNA is transcriptionally repressed by the presence of methionine in the growth medium. By primer extension the MET14 transcripts were found to start between positions −25 and −45 upstream of the initiator codon. Located upstream of the MET14 gene is a perfect match (positions −222 to −229) with the previously proposed methionine-specific upstream activating sequence (UASMet). This is the same as the consensus sequence of the Centromere DNA Element I (CDEI) that binds the Centromere Promoter Factor I (CPFI) and of two regulatory elements of the PH05 gene to which the yeast protein PH04 binds. The human oncogenic protein c-Myc also has the same recognition sequence. Furthermore, in the 270 by upstream of the MET14 coding region there are several matches with a methionine-specific upstream negative (URSMet) control element. The significance of these sequences was investigated using different upstream deletion mutations of the MET14 gene which were fused to the lacZ gene of E. coli and chromosomally integrated. We find that the methionine-specific UASMet and one of the URSMet lie in regions necessary for strong activation and weak repression of MET14 transcription, respectively. We propose that both types of control are exerted on MET14.

Sequences upstream of the STE6 gene required for its expression and regulation by the mating type locus in Saccharomyces cerevisiae.

Abstract: The STE6 gene of Saccharomyces cerevisiae is an a-specific gene; it is repressed in alpha cells by the alpha 2 product of the mating type locus. To study the role of sequences upstream of STE6 in its regulation and expression, we have determined the DNA sequence of the promoter region, identified the start sites for the STE6 transcript, and identified sequences governing its transcription. Deletions that remove DNA upstream of the STE6 gene were produced and assayed for effects on regulation and expression. The deletions defined two intervals upstream of the STE6 transcription initiation sites. One contains all or part of a negative element; the other contains all or part of a positive element. The negative element is required for repression of STE6 by alpha 2: deletions lacking this element express STE6 constitutively. Such deletions remove a 31-base-pair site, located 135 base pairs upstream of the first transcript start site, that is highly homologous to sites present in the upstream regions of four other genes repressed by alpha 2. These sites are presumably responsible for repression of the a-specific genes by alpha 2. The positive element (a putative upstream activation site) is required for expression of STE6. The deletions define the left boundary of the proposed upstream activation site. Sequence homologies between STE6 and other a-specific genes are found in this region and may mediate activation of this set of genes.