A 30-amino-acid segment of C/EBP, a newly discovered enhancer binding protein, shares notable sequence similarity with a segment of the cellular Myc transforming protein. Display of these respective amino acid sequences on an idealized alpha helix revealed a periodic repetition of leucine residues at every seventh position over a distance covering eight helical turns. The periodic array of at least four leucines was also noted in the sequences of the Fos and Jun transforming proteins, as well as that of the yeast gene regulatory protein, GCN4. The polypeptide segments containing these periodic arrays of leucine residues are proposed to exist in an alpha-helical conformation, and the leucine side chains extending from one alpha helix interdigitate with those displayed from a similar alpha helix of a second polypeptide, facilitating dimerization. This hypothetical structure is referred to as the "leucine zipper," and it may represent a characteristic property of a new category of DNA binding proteins.

https://science.sciencemag.org/content/sci/240/4860/1759.full.pdf

The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins

Zinc coordination, function, and structure of zinc enzymes and other proteins

Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain.

Foreign gene expression in yeast: a review.

Glucose and related sugars repress the transcription of genes encoding enzymes required for the utilization of alternative carbon sources; some of these genes are also repressed by other sugars such as galactose, and the process is known as catabolite repression. The different sugars produce signals which modify the conformation of certain proteins that, in turn, directly or through a regulatory cascade affect the expression of the genes subject to catabolite repression. These genes are not all controlled by a single set of regulatory proteins, but there are different circuits of repression for different groups of genes. However, the protein kinase Snf1/Cat1 is shared by the various circuits and is therefore a central element in the regulatory process. Snf1 is not operative in the presence of glucose, and preliminary evidence suggests that Snf1 is in a dephosphorylated state under these conditions. However, the enzymes that phosphorylate and dephosphorylate Snf1 have not been identified, and it is not known how the presence of glucose may affect their activity. What has been established is that Snf1 remains active in mutants lacking either the proteins Grr1/Cat80 or Hxk2 or the Glc7 complex, which functions as a protein phosphatase. One of the main roles of Snf1 is to relieve repression by the Mig1 complex, but it is also required for the operation of transcription factors such as Adr1 and possibly other factors that are still unidentified. Although our knowledge of catabolite repression is still very incomplete, it is possible in certain cases to propose a partial model of the way in which the different elements involved in catabolite repression may be integrated.

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Yeast Carbon Catabolite Repression

Classical yeast genetics coupled with the cloning of regulatory genes by complementation of function1,2 is a powerful means of identifying and isolating trans-acting regulatory elements3–6. One such regulatory gene is ADR1 which encodes a protein required for transcriptional activation of the glucose-repressible alcohol dehydrogenase (ADH2) gene7,8. We now report the nucleotide sequence of ADR1; it encodes a polypeptide chain of 1,323 amino acids, of which the amino-terminal 302 amino acids are sufficient to stimulate ADH2 transcription. This active amino-terminal region shows amino-acid sequence homology with the repetitive DNA-binding domain of TFIIIA9, an RNA polymerase III transcription factor of Xenopus laevis10. Similar domains are found in proteins encoded at the Kruppel11 and Serendipity12 loci of Drosophila melanogaster. We discuss the implications of this structural homology and suggest that a similar domain may exist in other yeast regulatory proteins such as those encoded by GAL4 (ref. 13) and PPR1 (ref. 14).

Sequence homology of the yeast regulatory protein ADR1 with Xenopus transcription factor TFIIIA

The best-understood protein structure involved in DNA binding is the helix-turn-helix motif. A second DNA-binding domain, the finger structure, has been proposed on the basis of sequence analysis, partial proteolysis and zinc content of Xenopus transcription factor TFIIIA. Other eukaryotic proteins were subsequently found to contain contiguous repeat units of the postulated finger motif. Each repeat unit contains thirty amino acids and is thought to bind a zinc atom using two cysteines and two histidines as ligands. The protein loop or finger between apparent zinc ligands is rich in DNA-binding residues and is thought to make specific contacts with DNA. The yeast protein ADR1, a positive regulator of transcription of the gene ADH2, contains two finger domains in a region of the protein required for transcriptional activation. Nineteen independently isolated adr1 mutations induced by hydroxylamine were found at nine different amino-acid positions, seven of which are in the two finger domains. All four mutations that altered invariant cysteine or histidine residues led to an adr1 null phenotype. Only one other mutation caused an adr1 null phenotype. Thus, one finger domain is not sufficient for ADR1 activity. This provides the first evidence that, as is consistent with the proposed model, the invariant cysteine and histidine residues are essential for the formation of the finger structure.

Two zinc fingers of a yeast regulatory protein shown by genetic evidence to be essential for its function

The yeast ADR1 protein contains two zinc finger domains that are essential for its role in transcriptional activation of alcohol dehydrogenase (ADH2). These domains are thought to function as DNA-binding structures. An ADR1-beta-galactosidase fusion protein made in Escherichia coli and containing the finger domains of ADR1 binds in vitro in a zinc-dependent manner to DNA fragments containing the two ADH2 upstream activation sequences. The strongest binding is to upstream activation sequence 1, a 22-base-pair palindrome.

The yeast regulatory protein ADR1 binds in a zinc-dependent manner to the upstream activating sequence of ADH2.

ADR1 is a transcription factor required for activation of the glucose-repressible alcohol dehydrogenase 2 (ADH2) gene in Saccharomyces cerevisiae. ADR1 has two zinc finger domains between amino acids 102 and 159, and it binds to an upstream activation sequence (UAS1) in the ADH2 promoter. A functional dissection of ADR1 was performed by using a series of amino- and carboxy-terminal deletion mutants of ADR1, most of which were fused to the Escherichia coli beta-galactosidase. These deletion mutants were assayed for binding to UAS1 in vitro, for the ability to activate ADH2 transcription in vivo, and for level of expression. Deletion of ADR1 amino acids 150 to 172 and 76 to 98 eliminated DNA binding in vitro, which accounted for the loss of transcriptional activation in vivo. Results with the former deletion mutant indicated that both of the ADR1 zinc fingers are necessary for sequence-specific DNA binding. Results with the latter deletion mutant suggested that at least part of the sequence between amino acids 76 to 98, in addition to the two finger domains, is required for high-affinity DNA binding. The smallest fusion protein able to activate ADH2 transcription, containing ADR1 amino acids 76 to 172, was much less active in vivo than was the longest fusion protein containing amino acids 1 to 642 of ADR1. In addition, multiple regions of the ADR1 polypeptide (including amino acids 40 to 76, 260 to 302, and 302 to 505), which are required for full activation of ADH2, were identified. An ADR1-beta-galactosidase fusion protein containing only the amino-terminal 16 amino acids of ADR1 was present at a much higher level than were larger fusion proteins, which suggested that the sequences within ADR1 influence the expression of the gene fusion.

Localization of a minimal binding domain and activation regions in yeast regulatory protein ADR1.

Disruption of ADR1, a positive regulatory gene in the yeast Saccharomyces cerevisiae, abolished derepression of ADH2 but did not affect glucose repression of ADH2 or cell viability. The ADR1 mRNA was 5 kilobases long and had an unusually long leader containing 509 nucleotides. ADR1 mRNA levels were regulated by the carbon source in a strain-dependent fashion. beta-Galactosidase levels measured in strains carrying an ADR1-lacZ gene fusion paralleled ADR1 and ADR1-lacZ mRNA levels, indicating a lack of translational regulation of ADR1 mRNA. ADH2 was regulated by the carbon source to the same extent in all strains examined and showed complete dependence on ADR1 as well. The expression of ADR1 mRNA and an ADR1-beta-galactosidase fusion protein during glucose repression suggested that the activity of the ADR1 protein is regulated at the posttranslational level to properly regulate ADH2 expression. The ADR1-beta-galactosidase fusion protein was able to activate ADH2 expression during glucose repression but showed significantly higher levels of activation upon derepression. A similar result was obtained when ADR1 was present on a multicopy plasmid. These results suggest that low-level expression of ADR1 is required to maintain glucose repression of ADH2 and are consistent with the hypothesis that ADR1 is regulated at the posttranslational level.

Hal Blumberg

Papers

Sequence homology of the yeast regulatory protein ADR1 with Xenopus transcription factor TFIIIA

Two zinc fingers of a yeast regulatory protein shown by genetic evidence to be essential for its function

The yeast regulatory protein ADR1 binds in a zinc-dependent manner to the upstream activating sequence of ADH2.

Localization of a minimal binding domain and activation regions in yeast regulatory protein ADR1.

Regulation of expression and activity of the yeast transcription factor ADR1.