Positive regulation of the beta-galactosidase gene from Kluyveromyces lactis is mediated by an upstream activation site that shows homology to the GAL upstream activation site of Saccharomyces cerevisiae.

Abstract

In contrast to the Escherichia coli lac operon, the yeast 1-galactosidase gene is positively regulated. In the 5'-noncoding region of the Kluyveromyces lactis LAC4 gene, we manped an upstream activation site (UAS) that is required for induction. This sequence, located between positions -435 and -326 from the start of translation, functions irrespective of its orientation and can confer lactose regulation to the heterologous CYC1 promoter. It is composed of at least two subsequences that must act in concert. One of these subsequences showed a strong homology to the UAS consensus sequence of the Saccharomyces cerevisiae GAL genes (E. Giniger, S. M. Varnum, and M. Ptashne, Cell 40:767-774, 1985). We propose that this region of homology located at about position -426 is a binding site for the product of the regulatory gene LAC9 which probably induces transcription of the LAC4 gene in a manner analogous to that of the GAL4 protein.The yeast Kluyveromyces lactis is a eucaryotic unicellular organism that can utilize lactose as a sole carbon source. It offers an interesting possibility in that the regulation of its LAC genes can be compared directly with the analogous lac operon of Escherichia coli. As is typical for eucaryotes these genes are not organized in an operon but are coordinately regulated (8,30). Both the LAC4 gene, encoding Igalactosidase (33), and the LAC12 gene, encoding lactose permease (34), are inducible by lactose and galactose. Their regulation is thereby closely coupled to that of the GAL genes. The lactose permease gene and the P-galactosidase gene have been cloned (9, 34) and have been shown to be tightly linked, mapping within 13 kilobase pairs (kbp) (34). The structural genes of the Leloir pathway enzymes GAL], GAL1O, and GAL7 (in analogy to the Saccharomyces cerevisiae genes) are also clustered and show the same gene order GAL7 GALIO GAL] as in S. cerevisiae (30).Recessive regulatory K. lactis mutants have been isolated (10, 32; M. Ruzzi and A. Walker-Jonah, unpublished data) in which expression of the LAC as well as the GAL genes (8; Ruzzi and Walker-Jonah, unpublished data) are affected. They fall into two classes: noninducible (lac9) and constitutive (lacJO) mutations (10, 32).In S. cerevisiae both the GAL genes and the MEL] gene, which encode a-galactosidase, are transcriptionally regulated by the products of the regulatory genes GAL4 and GAL80 (29; for a review, see reference 27). Recessive mutations in GAL4 are noninducible (11), and gal80 mutants are constitutive (12). It has been shown that the GAL4 protein binds to the 5'-noncoding regions of the genes regulated by galactose (2, 13), thereby activating transcription in the presence of galactose. Under noninducing conditions the GAL80 gene product probably inactivates the GAL4 protein (25,26,28 has been mapped (2, 13) and coincides with the GAL upstream activation sites (UASG) that were previously shown to be required for induction in cis (36).In K. lactis LAC4 is the best-characterized gene of lactose-galactose metabolism. The gene has...

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.

“…Deletion of the base pair reduced binding eightfold, a value not much different than that for LAC4UASI, a UAS that is necessary for normal function of the lactose-galactose regulon (3,13,18). In spite of our data, the center base pair must play an essential role in LAC9 binding or some other LAC9 activity, since all natural K. lactis UASs contain 17, not 16, bp (Table 1).…”

“…Transcriptional enhancement of the regulon is controlled so that in the absence of inducer there is low expression, while in the presence of an inducer, such as lactose (7), expression is high. In order to enhance transcription of the target genes, LAC9 must bind to a 17-bp element, termed an upstream activating sequence (UAS), having the consensus form 5'-CGG(N5)(A/T)(N5)CCG-3' (3,13,18). The UASs are located upstream of each gene in the regulon and are always grouped in two or four copies (summarized in reference 8).…”

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.

“…Since this initial report, many proteins have been shown to have one or more copies of a zinc finger with the general form Cys-X2 4-Cys-X2_j5-a-X2-4-a or a-X24-a-X2-15-Cys-X2 -Cys, where a is Cys or His (1). It remains to be determined how a single or multiple zinc fingers bind DNA.We are examining how a trans-acting, positive regulatory protein, the LAC9 protein of the yeast Kluyveromyces lactis, regulates transcription of five structural genes which constitute the lactose-galactose regulon (23,28 (16,25). It has been hypothesized that the transcriptional activator function of the LAC9 protein is modulated by the LAC10 protein.…”

“…We are examining how a trans-acting, positive regulatory protein, the LAC9 protein of the yeast Kluyveromyces lactis, regulates transcription of five structural genes which constitute the lactose-galactose regulon (23,28 (16,25). It has been hypothesized that the transcriptional activator function of the LAC9 protein is modulated by the LAC10 protein.…”

Crystal structure of a PPR1-DNA complex: DNA recognition by proteins containing a Zn2Cys6 binuclear cluster.

“…la; for review, see Johnston 1987;Andre 1990). DNA targets for several members of this family have two symmetrically placed CGG elements, separated by a spacing characteristic of the particular protein that recognizes the site (Bram and Romberg 1985;Giniger et al 1985;Breunig and Kuger 1987;Le onardo et al 1987;Ruzzi et al 1987;Friden and Schimmel 1988;Siddiqui and Brandriss 1989;Roy et al 1990;Fig. lb).…”