Characterization of the DNA-binding activity of GCR1: In vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae

Michael A. Huie, Edward W. Scott, Carolyn M. Drazinic, M. Cecilia Lopez, Ian K. Hornstra, Thomas P. Yang, Henry V. Baker

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Abstract

GCR1 gene function is required for high-level glycolytic gene expression in Saccharomyces cerevisiae. Recently, we suggested that the CTTCC sequence motif found in front of many genes encoding glycolytic enzymes lay at the core of the GCR1-binding site. Here we mapped the DNA-binding domain of GCR1 to the carboxy-terminal 154 amino acids of the polypeptide. DNase I protection studies showed that a hybrid MBP-GCR1 fusion protein protected a region of the upstream activating sequence of TPI (UASTPI), which harbored the CTTCC sequence motif, and suggested that the fusion protein might also interact with a region of the UAS that contained the related sequence CATCC. A series of in vivo G methylation protection experiments of the native TPI promoter were carried out with wild-type and gcr1 deletion mutant strains. The G doublets that correspond to the C doublets in each site were protected in the wild-type strain but not in the gcr1 mutant strain. These data demonstrate that the UAS of TPI contains two GCR1-binding sites which are occupied in vivo. Furthermore, adjacent RAP1/GRF1/TUF- and REB1/GRF2/QBP/Y-binding sites in UASTPI were occupied in the backgrounds of both strains. In addition, DNA band-shift assays were used to show that the MBP-GCR1 fusion protein was able to form nucleoprotein complexes with oligonucleotides that contained CTTCC sequence elements found in front of other glycolytic genes, namely, PGK, ENO1, PYK, and ADH1, all of which are dependent on GCR1 gene function for full expression. However, we were unable to detect specific interactions with CTTCC sequence elements found in front of the translational component genes TEF1, TEF2, and CRY1. Taken together, these experiments have allowed us to propose a consensus GCR1-binding site which is 5′-(T/A)N(T/C)N(G/A)NC(T/A)TCC(T/A)N(T/A)(T/A)(T/G)-3′.

Original languageEnglish (US)
Pages (from-to)2690-2700
Number of pages11
JournalMolecular and Cellular Biology
Volume12
Issue number6
StatePublished - Jun 1992
Externally publishedYes

Fingerprint

Saccharomyces cerevisiae
Binding Sites
DNA
Genes
Guanine Nucleotide-Releasing Factor 2
Gene Components
Proteins
Nucleoproteins
Deoxyribonuclease I
Oligonucleotides
Methylation
Gene Expression
Amino Acids
Peptides
Enzymes

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Huie, M. A., Scott, E. W., Drazinic, C. M., Lopez, M. C., Hornstra, I. K., Yang, T. P., & Baker, H. V. (1992). Characterization of the DNA-binding activity of GCR1: In vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae. Molecular and Cellular Biology, 12(6), 2690-2700.

Characterization of the DNA-binding activity of GCR1 : In vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae. / Huie, Michael A.; Scott, Edward W.; Drazinic, Carolyn M.; Lopez, M. Cecilia; Hornstra, Ian K.; Yang, Thomas P.; Baker, Henry V.

In: Molecular and Cellular Biology, Vol. 12, No. 6, 06.1992, p. 2690-2700.

Research output: Contribution to journalArticle

Huie, Michael A. ; Scott, Edward W. ; Drazinic, Carolyn M. ; Lopez, M. Cecilia ; Hornstra, Ian K. ; Yang, Thomas P. ; Baker, Henry V. / Characterization of the DNA-binding activity of GCR1 : In vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae. In: Molecular and Cellular Biology. 1992 ; Vol. 12, No. 6. pp. 2690-2700.
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abstract = "GCR1 gene function is required for high-level glycolytic gene expression in Saccharomyces cerevisiae. Recently, we suggested that the CTTCC sequence motif found in front of many genes encoding glycolytic enzymes lay at the core of the GCR1-binding site. Here we mapped the DNA-binding domain of GCR1 to the carboxy-terminal 154 amino acids of the polypeptide. DNase I protection studies showed that a hybrid MBP-GCR1 fusion protein protected a region of the upstream activating sequence of TPI (UASTPI), which harbored the CTTCC sequence motif, and suggested that the fusion protein might also interact with a region of the UAS that contained the related sequence CATCC. A series of in vivo G methylation protection experiments of the native TPI promoter were carried out with wild-type and gcr1 deletion mutant strains. The G doublets that correspond to the C doublets in each site were protected in the wild-type strain but not in the gcr1 mutant strain. These data demonstrate that the UAS of TPI contains two GCR1-binding sites which are occupied in vivo. Furthermore, adjacent RAP1/GRF1/TUF- and REB1/GRF2/QBP/Y-binding sites in UASTPI were occupied in the backgrounds of both strains. In addition, DNA band-shift assays were used to show that the MBP-GCR1 fusion protein was able to form nucleoprotein complexes with oligonucleotides that contained CTTCC sequence elements found in front of other glycolytic genes, namely, PGK, ENO1, PYK, and ADH1, all of which are dependent on GCR1 gene function for full expression. However, we were unable to detect specific interactions with CTTCC sequence elements found in front of the translational component genes TEF1, TEF2, and CRY1. Taken together, these experiments have allowed us to propose a consensus GCR1-binding site which is 5′-(T/A)N(T/C)N(G/A)NC(T/A)TCC(T/A)N(T/A)(T/A)(T/G)-3′.",
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