The DNA and protein factors which regulate the expression of eukaryotic genes are being studied at multiple levels, using the yeast Saccharomyces cerevisiae. This simple, single-celled eukaryote offers extensive classical genetic approaches, all of the advantages of recombinant DNA technology, and the simplicity of a eukaryotic microbe.
The genes under study encode isoenzymes involved in alcohol metabolism, a small family of highly homologous alcohol dehydrogenase (ADH) enzymes which differ in physiological function, cellular location, and genetic regulation. The intracellular concentration of these enzymes is controlled at the transcriptional level by metabolites, regulatory proteins, and cis-acting DNA sequences. Identification of the DNA sequences mediating expression and regulation of these genes has been accomplished by isolating and characterizing mutants with altered gene expression. Exact gene replacement allows new combinations of genetic elements to be tested in their correct chromosomal environment.
Dr. Young's group has cloned the genes encoding several transcription factors essential for expression of the genes under stringent metabolic control. One of these, Adr1, is a member of the class of proteins whose DNA-binding domain is dependent on Zn2+. The structure of the DNA binding domain of this protein was determined by high resolution NMR in collaboration with Dr. Rachel Klevit. This factor binds DNA in a novel way: Two monomers independently bind to adjacent sites with dyad symmetry. DNA binding in vivo is inhibited by nucleosomes with de-acetylated histone tails. Conversely, binding is permitted when the histone tails are hyper-acetylated. The activity of the transcription factor is regulated in a novel manner by the AMP-activated protein kinase, AMPK, or Snf1 as it is known in yeast. Phosphorylation of the transcription factor inactivates its ability to interact with coactivators because it binds ubiquitous 14-3-3 proteins. Dephosphorylation activates the transcription factor, releasing it from 14-3-3-mediated inhibition. Adr1 is an example of a transcription factor whose activity is highly regulated by redundant and over-lapping mechanisms, ensuring that transcription of downstream target genes occurs in a tightly controlled manner.
