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.

Regulation of gene expression and adhesion in Saccharomyces cerevisiae

Abstract: Adherence represents one important and initial virulence factor of fungal pathogenicity In the model fungus Saccharomyces cerevisiae adherence to substrates or to other cells depends on nutrients and is part of complex developmental processes, such as haploid invasive growth or diploid pseudohyphal formation Adherence per se can also be induced by amino acid starvation This specific adaptation requires the adhesin Flo11p and the transcriptional activator of the general amino acid control system Gcn4p A genome-wide transcriptional analysis of ∑1278b yeast cells under adhesion-inducing conditions imposed by amino acid starvation was performed to identify specifically regulated genes 22 novel genes were inducible by amino acid starvation 72 genes of different functional groups showed a previously unrecognized dependence upon Gcn4p under adhesion-inducing conditions In addition, several genes were identified as inducible by amino acid starvation in a Gcn4p-independent manner 2D-DIGE experiments of ∑1278b yeast cells were carried out to identify regulated proteins under adhesion-inducing conditions Seven protein spots displayed a highly increased intensity in response to amino acid starvation These protein spots were identified by mass spectrometry as Cpc2p, Efb1p, His1p, Hsp60p, Sod1p, Tpi1p and Tpm1p Comparisons with the respective transcriptional profiles revealed that the mRNA levels of the encoding genes were significantly increased only for the HIS1 gene Deletion of CPC2, which encodes a highly conserved G?-like WD-repeat protein, results in an adhesion deficient phenotype of amino acid-starved yeast cells CPC2 is also required for basal expression and activation of FLO11 under amino acid starvation The adherence-dependent developmental processes of haploid invasive growth and diploid pseudohyphal formation also depend on CPC2 During utilization of the fermentable carbon source glucose, transcription of CPC2 is induced CPC2 promoter analyses were performed to analyse regulation, and identified two upstream activation sequence elements required for basal expression and regulation of CPC2 The forkhead-like transcription factor Fhl1p and its co-factor Ifh1p were found as trans-acting elements Deletion of FHL1 reduces CPC2 transcription significantly in presence of glucose, whereas increased amounts of Ifh1p induces CPC2 transcription even under utilization of the non-fermentable carbon source ethanol

Investigation of the mechanism of ADR1 mediated transcriptional activation

Abstract: The purpose of these studies was to examine the mechanism of function of the transcriptional activator ADR1 of yeast Saccharomyces cerevisiae. ADR1 is required for the activation of the ADH2 gene, as well as genes involved in glycerol metabolism and peroxisome biogenesis. ADR1 is a DNA-binding protein. It binds to a 22 bp palindromic upstream activating sequence 1 (UAS1) element located 215 bp 5$\sp\prime$ to the transcription start site of ADH2. Elements similar to UAS 1 are found in promoters of all ADR1-dependent genes. ADR1 was shown previously to contain three transcriptional activation domains (TADs): TADI (amino acids 76-172), TADII (263-357) and TADIII (420-462). A novel activation domain, TADIV (residues 642-704), was identified in ADR1. Analysis of different derivatives of TADIV indicated that its activation function is principally localised to residues 698-704. In contrast to deletion of other ADR1 activation domains, deletion of activation domain TV from ADR1 severely compromised its ability to activate ADH2 transcription. ADR1 activation domains have been shown to directly interact in vitro with ADA2 and GCN5 components of the ADA2 co-activator complex, and both ADA2 and GCN5 are also required for full ADH2 derepression. In addition, direct interaction of ADR1 TADs with TFIIB was observed, in which TADI displayed the strongest binding. A point mutation in TFIIB that reduced ADH2 derepression was found to result in decreased in vitro interaction of TFIIB with TADI. These results suggest that TFIIC is a functional contact for ADR1 activation of transcription. Defects in ADA2, GCN5 and TFIIB do not severely reduce ADR1 ability to activate transcription, suggesting that there may be additional contacts that ADR1 makes with the components of transcriptional machinery. In order to identify these proteins, I examined which known components of the general transcription machinery are retained by the ADR1 activation domains from yeast whole cell extracts. It was found that ADR1 transcription activation domain IV (TADIV) specifically retained core transcription factor IID. Moreover, ADR1 could be co-immunoprecipitated with the TAF90 component of the TFIID from yeast whole cell extracts. ADH2 activation by ADR1 required the presence of intact TFIID in vivo, suggesting that the physical interactions that were observed had functional relevance. We propose that multiple contacts made by the ADR1 activation domains to different components of the general transcriptional machinery and to the ADA2 co-activator complex contribute synergistically to the activation of the ADH2 gene. After facilitating the first rate limiting step – for example, TFIID recruitment, other steps in the preinitiation complex assembly become rate limiting, and even though transcription rate may have already become significant, it is further increased by ADR1 establishing new contacts to TFIIB and possibly other general transcription factors.

Enhanced protein production method

Abstract: An expression vector comprising a promoter, a coding sequence of a heterologous protein, the coding sequence being operably linked to the promoter, and an intronic sequence downstream of the promoter and upstream of the coding sequence, the intronic sequence comprising two identical donor sites and one acceptor site.