Transcriptional regulation of ribosomal protein genes in yeast secretory mutants

(1)构建了一系列含有CYH2上游区的融合基因，除Rap1p结合位点外，其余区域均有缺失，与LacZ基因融合。每个被检测到转录的融合基因都受到分泌途径缺陷的抑制。因此，我一直无法将UAS中的阳性元件与对sec突变作出反应的抑制元件分开，并认为这种抑制是通过Rap1p进行的。基于凝胶位移分析，我提出了一种假设，即Rap1p的修饰或未知调控因子(S)与Rap1p的结合是导致转录受阻的原因。(2)Rap1p不仅控制核糖体蛋白基因的转录，还控制糖酵解基因的转录。然而，核糖体蛋白基因的转录在分泌途径的缺陷下被特异性地抑制。糖酵解基因的转录也受Gcr1p的调控。我用部分CYH2启动子和部分PGK启动子构建了多种嵌合启动子。在限制性温度下，将Gcr1p位点插入到Rap1p位点附近会显著抑制转录抑制。由于没有Rap1p的Gcr1p即使在允许的温度下也只能轻微地激活转录，因此我们认为Gcr1p在限制性温度下不激活转录，但Gcr1p保护Rap1p免受其他蛋白的修饰或结合(S)。(3)为了从酵母突变株中分离出即使在限制性温度下核糖体蛋白基因的转录也不受抑制的双突变株，我构建了一个含有CyH2启动子区域的融合基因，将其与编码短命β-半乳糖苷酶的突变LacZ融合。我现在正在筛选双重变种人。

(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.