IDENTIFICATION OF YEAST PKA SUBUNITS TRANSCRIPTION REGULATORS USING TWO-COLOR CELL ARRAY SCREENING

PAUTASSO, CONSTANZA; ZAREMBERG, VANINA; ROSSI, SILVIA

Potrero de los Funes, San Luis

Congreso; XLVII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2011

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Transcriptional regulation is a key mechanism that controls the fate and response of cells to diverse signals. Therefore, the identification of the DNA-binding proteins, which mediate these signals, is a crucial step in elucidating how cell fate is regulated. We applied a functional genomic approach to scrutinize the promoters of the yeast PKA subunits: TPKs and BCY, to contribute to the understanding of the specificity of cAMP-PKA pathway. We have used a two-color GFP-RFP reporter system: Reporter-Synthetic Genetic Array to systematically assess the effects of gene deletions on the TPK1, TPK2 TPK3 and BCY1 promoter activities. The screen allows measurement of a promoter?GFP reporter gene and a control promoter?RFP reporter gene in an array of yeast deletion mutants and provides quantitative measures of promoter gene activity in each mutant background. We identified potential transcription factors, many of which have no perfect consensus site within the promoters and also that transcription of these genes are repressed by the PKA activity itself, and also in presence of glucose, but not in presence of a nonfermentable carbon source.Our analysis also supports the view that although comparative analysis can provide a useful guide, functional assays are required for accurate identification of TF-binding site interactions in complex promoters