Transcriptional regulation of ribosomal protein genes in yeast secretory mutants

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(1) I have constructed a series of fusion genes containing the CYH2 upstream region, with various deletions except a Rap1p binding site, fused to lacZ gene. Each fusion gene for which transcription is detected is subject to repression by the defect in the secretory pathway. Thus I have been unable to separate the positive element in the UAS from the repressive element responding to sec mutations and it is suggested that the repression is through Rap1p. Based on gel-shift assay, I propose a hypothesis that the modification of Rap1p or the binding of unknown regulatory factor (s) to Rap1p is responsible to the repression of transcription.(2) Rap1p controls the transcription of not only ribosomal protein genes but also glycolytic genes. However, the transcription of ribosomal protein genes is repressed specifically under the defect in secretory pathway. The transcription of the glycolytic genes is also controled by Gcr1p. I have constructed various kinds of chimeric promoter with a part of CYH2 promoter and a part of PGK promoter. The insertion of a Gcr1p site close to a Rap1p site caused the significant suppression of the transcriptional repression at the restrictive temperature. As Gcr1p without Rap1p activates the transcription only slightly even at the permissive temperature, it is suggested that Gcr1p dose not activate the transcription instead of Rap1p at the restrictive temperature, but that Gcr1p protects Rap1p from the modification or the binding of other protein (s).(3) In order to isolate double mutants from yeast ts sec mutant strains, in which the transcription of ribosomal protein genes is not repressed even at the restrictive temperature, I have constructed a fusion gene containing promoter region of CYH2 fused to mutagenized lacZ encoding short-lived beta-galactosidase. I am now screening for the double mutants.