Hyen Sam Kang

Nrg1 Is a Transcriptional Repressor for Glucose Repression of STA1 Gene Expression in Saccharomyces cerevisiae

ABSTRACT Expression of genes encoding starch-degrading enzymes is regulated by glucose repression in the yeast Saccharomyces cerevisiae. We have identified a transcriptional repressor, Nrg1, in a genetic screen designed to reveal negative factors involved in the expression of STA1, which encodes a glucoamylase. TheNRG1 gene encodes a 25-kDa C2H2zinc finger protein which specifically binds to two regions in the upstream activation sequence of the STA1 gene, as judged by gel retardation and DNase I footprinting analyses. Disruption of theNRG1 gene causes a fivefold increase in the level of theSTA1 transcript in the presence of glucose. The expression of NRG1 itself is inhibited in the absence of glucose. DNA-bound LexA-Nrg1 represses transcription of a target gene 10.7-fold in a glucose-dependent manner, and this repression is abolished in bothssn6 and tup1 mutants. Two-hybrid and glutathione S-transferase pull-down experiments show an interaction of Nrg1 with Ssn6 both in vivo and in vitro. These findings indicate that Nrg1 acts as a DNA-binding repressor and mediates glucose repression of the STA1 gene expression by recruiting the Ssn6-Tup1 complex.

Inactivation of the UASI of STA1 by glucose and STA10 and identification of two loci, SNS1 and MSS1, involved in STA10-dependent repression in Saccharomyces cerevisiae

It has been reported that two upstream activation sites, UASI and UAS2, exist in the 5′ non-coding region of the STA1 gene of Saccharomyces cerevisiae var. diastaticus. Based on studies using a UAS1STA1-CYCI-lacZ fusion, we divided UASI into two subsites, UASI-1 and UASI-2. The activation of the CYC1 promoter by UAS1STA1 was repressed by glucose in the culture medium and by the STA10 gene. The MATa/MATα mating type configuration did not, however, affect UAS1STA1 activation. The UAS1STA1-CYC1-lacZ expression system was used to study STA10 repression further. A mutant insensitive to STA10-dependent repression was isolated. This sns1 mutation was not linked to STA10 and partially overcame the repressive effect of STA10 at the transcriptional level. From a genomic library constructed in the UAS1STA1-CYC1-lacZ expression vector, the MSS1 locus (multicopy suppressor of sns1) was isolated. This suppression of the sns1 mutation by multiple copies of the mss1 locus occurred at the transcriptional level. When a gene disruption experiment was performed to examine the effect of a mss1 mutation, the sns1 mss1 double mutants produced 4 times higher levels of STA1 transcripts in the presence of STA10 than did the sns1 strain. Data presented in this paper suggest that both SNS1 and MSS1 loci are involved in STA10-dependent repression.

ISOLATION OF AN EXTRAGENIC SUPPRESSOR OF THE RNA1-1 MUTATION IN SACCHAROMYCES CEREVISIAE

Abstract The small GTPase Ran is essential for nucleocytoplasmic transport of macromolecules. In the yeast Saccharomyces cerevisiae, Rna1p functions as a Ran-GTPase activating protein (RanGAP1). Strains carrying the rna1-1 mutation exhibit defects in nuclear transport and, as a consequence, accumulate precursor tRNAs. We have isolated two recessive suppressors of the rna1-1 mutation. Further characterization of one of the suppressor mutations, srn10-1, reveals that the mutation (i) can not bypass the need for Rna1p function and (ii) suppresses the accumulation of unspliced pre-tRNA caused by rna1-1. The SRN10 gene is not essential for cell viability and encodes an acidic protein (pI = 5.27) of 24.8 kDa. Srn10p is located in the cytoplasm, as determined by indirect immunofluorescence microscopy. Two-hybrid analysis reveals that there is a physical interaction between Srn10p and Rna1p in vivo. Our results identify a protein that interacts with the yeast RanGAP1.