Characterization of TPI gene expression in isogeneic wild-type and gcr1-deletion mutant strains of Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae the enzymes of glycolysis constitute 30-60 percent of the soluble protein. GCR1 gene function is required for high level glycolytic gene expression. We have undertaken a biochemical and genetic characterization of TPI, a gene affected by gcr1 lesions. Northern analysis showed that steady-state levels of TPI transcripts are severely reduced in gcr1 mutant strains. However, primer extension experiments revealed that TPI transcripts isolated from wild-type and gcr1 mutant strains have identical 5' ends. To map the 5' boundary of TPI controlling region, we employed a TPI::lacZ gene fusion carrying 3.5 kb 5' to the translational start of the TPI structural gene. Nuclease Bal31 deletion analysis demonstrated that sequences sufficient for high level expression of TPI reside within 392 nucleotides preceding the start of the structural gene. We have identified GRF1/RAPI/TUF-binding site positioned 339 to 349 bp 5' to the translation start of TPI. DNA band shift assays were carried out with wild-type and gcr1 deletion mutant strains, and similar patterns of band shifting were observed.