The goal is to understand the molecular mechanism of transcriptional regulation in a eucaryote. As a model system we are studying the inducible lactose-galactose regulon in the yeast Kluyveromyces lactis. We have isolated LAC9, a trans-acting positive regulatory gene, and shown that it has both similarities and differences to the GAL4 regulatory gene of S. cerevisiae. This relationship provides a unique opportunity to understand how the LAC9 protein functions, how it connects to the induction signal, and how it connects to global regulatory circuits including glucose repression.
Our specific aims are to (1) determine which amino acids in the LAC9 protein specify binding to Upstream Activator Sites (UAS), (2) determine which domains of LAC9 protein sense the induction signal, mediate glucose repression, and activated transcription, (3) purify the LAC9 protein and characterize its interaction with UAS, (4) use purified LAC9 protein to identify and characterize interactions with other proteins, (5) determine if there are cis-acting promoter elements besides UAS and how they function, (6) determine which bases in the 17 bp UAS specify binding of LAC9 protein, (7) examine the hypothesis that LAC10 protein acts as a negative regulator by binding directly to LAC9 protein. Recombinant DNA techniques will be used to isolate genes and change their base sequence, and biochemical techniques will used to study DNA-protein and protein-protein interactions. The information obtained from these studies will aid our understanding of more complex regulatory phenomena including cellular differentiation, environmental adaptation, and disease states such as cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM022749-14
Application #
3271300
Study Section
Biochemistry Study Section (BIO)
Project Start
1979-04-01
Project End
1992-03-30
Budget Start
1989-04-01
Budget End
1990-03-31
Support Year
14
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Kentucky
Department
Type
Schools of Medicine
DUNS #
832127323
City
Lexington
State
KY
Country
United States
Zip Code
40506
Czyz, M; Nagiec, M M; Dickson, R C (1993) Autoregulation of GAL4 transcription is essential for rapid growth of Kluyveromyces lactis on lactose and galactose. Nucleic Acids Res 21:4378-82
Kuzhandaivelu, N; Jones, W K; Martin, A K et al. (1992) The signal for glucose repression of the lactose-galactose regulon is amplified through subtle modulation of transcription of the Kluyveromyces lactis Kl-GAL4 activator gene. Mol Cell Biol 12:1924-31
Mylin, L M; Gerardot, C J; Hopper, J E et al. (1991) Sequence conservation in the Saccharomyces and Kluveromyces GAL11 transcription activators suggests functional domains. Nucleic Acids Res 19:5345-50
Halvorsen, Y D; Nandabalan, K; Dickson, R C (1991) Identification of base and backbone contacts used for DNA sequence recognition and high-affinity binding by LAC9, a transcription activator containing a C6 zinc finger. Mol Cell Biol 11:1777-84
Pan, T; Halvorsen, Y D; Dickson, R C et al. (1990) The transcription factor LAC9 from Kluyveromyces lactis-like GAL4 from Saccharomyces cerevisiae forms a Zn(II)2Cys6 binuclear cluster. J Biol Chem 265:21427-9
Witte, M M; Dickson, R C (1990) The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1. Mol Cell Biol 10:5128-37
Dickson, R C; Gerardot, C J; Martin, A K (1990) Genetic evidence for similar negative regulatory domains in the yeast transcription activators GAL4 and LAC9. Nucleic Acids Res 18:5213-7
Halvorsen, Y C; Nandabalan, K; Dickson, R C (1990) LAC9 DNA-binding domain coordinates two zinc atoms per monomer and contacts DNA as a dimer. J Biol Chem 265:13283-9
Webster, T D; Dickson, R C (1988) The organization and transcription of the galactose gene cluster of Kluyveromyces lactis. Nucleic Acids Res 16:8011-28
Witte, M M; Dickson, R C (1988) Cysteine residues in the zinc finger and amino acids adjacent to the finger are necessary for DNA binding by the LAC9 regulatory protein of Kluyveromyces lactis. Mol Cell Biol 8:3726-33

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