When regulation of GCR1 expression was analysed using a GCR1-lacZ fusion, lacZ expression levels were decreased in the Deltagcr1 or Deltagcr2 mutant. RT-PCR analysis of genomic GCR1 transcript confirmed the dependency of GCR1 expression on the Gcr1p-Gcr2p complex. Examination of the 5' non-coding region of GCR1 identified three putative Gcr1p binding sites (CT-boxes) in the -100 to -200 region of GCR1, and the putative binding sites for Rap1p (RPG-box) and Abf1p were also identified nearby. The region containing putative cis-elements was analysed by cloning it upstream of the CYC1TATA-lacZ fusion. The GCR1(UAS)-CYC1TATA-lacZ fusion showed a moderate activity and, as expected, the activity was drastically reduced in the Deltagcr1 or Deltagcr2 mutant. Systematic deletion and mutation analyses of cis-elements in this region demonstrated that the putative binding sites for Rap1p and Abf1p were not involved in the promoter activity of GCR1(UAS) and only one of the three CT-boxes showed GCR1- and GCR2-dependent promoter activity. In contrast to the expression of glycolytic genes, where a RPG-box adjacent to the CT-box is required for strong promoter activities, CT-box-dependent expression of GCR1 did not require the RPG-box. Also, a contribution of Sgc1p, an E-box binding transcription factor, to the expression of GCR1 was suggested, based on its disruption analysis.
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