Transcriptional regulation of ribosomal protein genes in yeast secretory mutants

(1)构建了一系列包含CYH2上游区域的融合基因，除了Rap1p结合位点存在多种缺失外，与lacZ基因融合。检测到转录的每个融合基因都受到分泌途径缺陷的抑制。因此，我无法将UAS中的阳性元件与响应sec突变的抑制元件分开，并且认为抑制是通过Rap1p进行的。基于凝胶转移实验，我提出了一个假设，Rap1p的修饰或未知调节因子与Rap1p的结合是抑制转录的原因。(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缺陷下被特异性抑制。糖酵解基因的转录也受Gcr1p的控制。我用一部分CYH2启动子和一部分PGK启动子构建了多种嵌合启动子。在限制性温度下，Gcr1p位点靠近Rap1p位点的插入导致转录抑制显著抑制。由于不含Rap1p的Gcr1p即使在允许温度下也只能轻微激活转录，提示Gcr1p在限制温度下也不会代替Rap1p激活转录，而是保护Rap1p不被修饰或与其他蛋白结合。(3)为了从酵母ts - sec突变株中分离出即使在限制温度下核糖体蛋白基因转录也不被抑制的双突变体，我构建了一个包含CYH2启动子区域的融合基因，该基因与突变的lacZ融合，编码短寿命的β -半乳糖苷酶。我现在正在筛查双突变体。

(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.