Abstract

Saccharomyces cerevisiae olumsuz çevre koşullarında stres metaboliti olarak trehaloz biriktirir. Hücre içi trehaloz miktarının düzenlenmesinde trehalozun sentezi ve yıkımı önemlidir. Bu nedenle, TPS1 ve NTH1 gen ekspresyonları transkripsiyon ve translasyon sırasında sıkı bir şekilde düzenlenmektedir. Her iki genin promotor bölgesinde Stres Tepki Elementleri (STRE) bulunduğundan stres koşullarında birlikte aktive olurlar. Ancak her iki genin promotor bölgesinde benzer düzenleyici elemanların bulunması bu genlerin transkripsiyon seviyesinde farklı bir regülasyona uğradıklarını göstermektedir. Çalışmamızda, TPS1 ve NTH1 genlerinin transkripsiyonel düzenlenmesinde Spt-Ada-Gcn5 Asetiltransferaz (SAGA) kompleksinin rolü besin yönünden zayıf ortamda belirlendi. Bu amaçla, SAGA kompleksinin alt ünitesi olan Ada1p içeren yaban tip ve içermeyen Δada1 mutant maya hücreleri azot açlığında ve normal büyüme koşullarında üretildi. Ayrıca yaban tip ve mutant maya hücrelerinde trehaloz seviyesi enzimatik olarak tespit edildi. TPS1 ve NTH1 genlerinin in silico promotor analizi sonucunda Msn2/4 transkripsiyon faktörlerinin bağlanması için gerekli olan STRE dizilerinin NTH1 promotorunda nükleozomlar tarafından kapatıldığı TPS1 promotorunda ise açıkta kaldıkları belirlendi. Ada1 proteininin yokluğunda, TPS1 geninde stres kaynaklı promotor aktivasyonu gözlenirken, NTH1 geninde promotor aktivasyonu gözlenmedi. Bu sonuçlara göre, Ada1 proteininin yokluğunda STRE dizilerini kaplayan nükleozomlar mobilize edilemediğinden Msn2/4 transkripsiyon faktörleri stres koşullarında promotora bağlanamayarak transkripsiyonu aktive edememiş olabilir. Ayrıca stres koşullarından bağımsız olarak Ada1 proteininin yokluğunda maya hücrelerindeki trehaloz birikiminin azaldığı gözlendi.

THE SAGA COMPLEX IS ESSENTIAL FOR THE REGULATION OF GENES INVOLVED IN YEAST TREHALOSE METABOLISM

Abstract

Saccharomyces cerevisiae accumulates trehalose as a stress metabolite in adverse environmental conditions. The trehalose synthesis and breakdown are important for the regulation of trehalose levels within the yeast cell. Therefore, TPS1 and NTH1 gene expressions are tightly regulated during transcription and also translation. Since both genes contain Stress Response Elements (STRE) in the promoter regions, they are co-activated under stress conditions. However, the presence of similar regulatory elements in the promoter of both genes shows that these genes undergo a different regulation at the transcriptional level. In our study, the role of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex in the transcriptional regulation of TPS1 and NTH1 genes was determined in nutrient-poor environment. For that purpose, the wild type and Δada1 mutant yeast cells, where Ada1p is a member of the SAGA complex, were grown in normal and nitrogen starvation conditions. In addition, trehalose level was detected enzymatically in both wild type and mutant yeast cells. In silico promoter analysis of TPS1 and NTH1 promoters revealed that the STRE sequences required for binding of Msn2/4 transcription factors are closed by nucleosomes at the NTH1 promoter, but open at the TPS1 promoter. In the absence of Ada1p, stress-induced promoter activation in the TPS1 gene was observed, while NTH1 gene expression was not activated. According to these results, the nucleosomes spanning the STRE sequences could not be mobilized in the absence of Ada1 protein, and therefore the Msn2/4 transcription factors cannot bind to the promoter and activate the NTH1 gene expression under stress conditions. It was also observed that in the absence of Ada1p, trehalose accumulation was reduced regardless of stress conditions. 

Keywords

• Msn2/4
• Nitrogen starvation
• Nucleosome
• SAGA
• Trehalose

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