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.

Interdependent recruitment of SAGA and Srb mediator by transcriptional activator Gcn4p.

Abstract: Transcriptional activation by Gcn4p is enhanced by the coactivators SWI/SNF, SAGA, and Srb mediator, which stimulate recruitment of TATA binding protein (TBP) and polymerase II to target promoters. We show that wild-type recruitment of SAGA by Gcn4p is dependent on mediator but independent of SWI/SNF function at three different promoters. Recruitment of mediator is also independent of SWI/SNF but is enhanced by SAGA at a subset of Gcn4p target genes. Recruitment of all three coactivators to ARG1 is independent of the TATA element and preinitiation complex formation, whereas efficient recruitment of the general transcription factors requires the TATA box. We propose an activation pathway involving interdependent recruitment of SAGA and Srb mediator to the upstream activation sequence, enabling SWI/SNF recruitment and the binding of TBP and other general factors to the promoter. We also found that high-level recruitment of Tra1p and other SAGA subunits is independent of the Ada2p/Ada3p/Gcn5p histone acetyltransferase module but requires Spt3p in addition to subunits required for SAGA integrity. Thus, while Tra1p can bind directly to Gcn4p in vitro, it requires other SAGA subunits for efficient recruitment in vivo.

Positive and negative elements regulate human interleukin 3 expression.

Abstract: The human interleukin 3 (IL-3) promoter is comprised of several cis-acting DNA sequences that modulate T-cell expression of IL-3. These are located within 315 nucleotides upstream of the mRNA start site. Transient expression of reporter genes linked to serially deleted sequences of the IL-3 promoter has allowed mapping of two activator sequences and an interposed repressor sequence. The proximal regulatory region is specific to IL-3 and prerequisite for efficient transcription. Its effect is enhanced by a second, more distal activating sequence consisting of an AP-1 binding site. Between the two activators lies a transcriptional silencer, which is a potent repressor in the absence of the AP-1 site. DNA-nuclear protein binding experiments demonstrate specific complex formation within each of these functional regions. Thus, both positive and negative regulatory elements appear to control expression of the human IL-3 gene in activated T cells.

Transcription regulation by murine B-myb is distinct from that by c-myb

Abstract: The transcription regulatory properties of murine B-myb protein were compared to those of c-myb. Whereas c-Myb trans-activated an SV40 early promoter containing multiple copies of an upstream c-Myb DNA-binding site (MBS-1), and similarly the human c-myc promoter, B-Myb was unable to do so. Full-length B-Myb translated in vitro did not bind MBS-1; however, truncation of the B-Myb C-terminus or fusion of the B-Myb DNA-binding domain to the c-Myb C-terminus showed that it was inherently competent to interact with this motif. Further evidence from co-transfection experiments, demonstrating that B-Myb inhibited trans-activation by c-Myb, suggested that failure of B-Myb to trans-activate these promoters did not simply occur through lack of binding to MBS-1. Moreover, using GAL4/B-Myb fusions, it was found that an acidic region of B-Myb, which by comparison to c-Myb was expected to contain a transcription activation domain, actually had no inherent trans-activation activity and indeed appeared to trans-inhibit c-Myb. In contrast to the above findings, both B-Myb and c-Myb were able to weakly trans-activate the DNA polymerase alpha promoter. Results obtained here demonstrate that the activities of B-Myb and c-Myb are clearly distinct and suggest that these related proteins may have different functions in regulation of target gene expression.

Saturation mutagenesis of an Escherichia coli rRNA promoter and initial characterization of promoter variants.

Abstract: Using oligonucleotide synthesis techniques, we generated Escherichia coli rrnB P1 (rrnB1p according to the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoter fragments containing single base substitutions, insertions, deletions, and multiple mutations, covering the whole length of the promoter including the upstream activation sequence (UAS). The activities of 112 mutant promoters were assayed as operon fusions to lacZ in lambda lysogens. The activities of most mutants with changes in the core promoter recognition region (i.e., substitutions, insertions, or deletions in the region of the promoter spanning the -10 and -35 E. coli consensus hexamers) correlated with changes toward or away from the consensus in the hexamer sequences or in the spacing between them. However, changes at some positions in the core promoter region not normally associated with transcriptional activity in other systems also had significant effects on rrnB P1. Since rRNA promoter activity varies with cellular growth rate, changes in activity can be the result of changes in promoter strength or of alterations in the regulation of the promoter. The accompanying paper (R. R. Dickson, T. Gaal, H. A. deBoer, P. L. deHaseth, and R. L. Gourse, J. Bacteriol. 171:4862-4870, 1989) distinguishes between these two alternatives. Several mutations in the UAS resulted in two- to fivefold reductions in activity. However, two mutants with changes just upstream of the -35 hexamer in constructs containing the UAS had activities 20- to 100-fold lower than the wild-type level. This collection of mutant rRNA promoters should serve as an important resource in the characterization of the mechanisms responsible for upstream activation and growth rate-dependent regulation of rRNA transcription.

A positive regulator of mitosis, Sok2, functions as a negative regulator of meiosis in Saccharomyces cerevisiae.

Abstract: The choice between meiosis and alternative developmental pathways in budding yeast depends on the expression and activity of transcriptional activator Ime1. The transcription of IME1 is repressed in the presence of glucose, and a low basal level of IME1 RNA is observed in vegetative cultures with acetate as the sole carbon source. IREu, a 32-bp element in the IME1 promoter, exhibits upstream activation sequence activity depending on Msn2 and -4 and the presence of acetate. We show that in the presence of glucose IREu functions as a negative element and that Sok2 mediates this repression activity. We show that Sok2 associates with Msn2. Sok2 functions as a general repressor whose availability and activity depend on glucose. The activity of Sok2 as a repressor depends on phosphorylation of T598 by protein kinase A (PKA). Relief of repression of Sok2 depends on both the N-terminal domain of Sok2 and Ime1. In the absence of glucose and the presence of Ime1 Sok2 is converted to a weak activator. Overexpression of Sok2 or mild expression of Sok2 with its N-terminal domain deleted leads to a decrease in sporulation. Previously it was reported that overexpression of Sok2 suppresses the growth defect resulting from a temperature-sensitive PKA; thus Sok2 has a positive role in mitosis. We show that Candida albicans Efg1, a homolog of Sok2, complements sok2Δ in repressing IREu. Our results demonstrate that Sok2, a positive regulator of mitosis, and Efg1, a positive regulator of filamentation, function as negative regulators of meiosis. We suggest that cells use the same regulators with opposing effects to ensure that meiosis will be an alternative to mitosis.