Characterization of a positive regulatory gene, LAC9, that controls induction of the lactose-galactose regulon of Kluyveromyces lactis: structural and functional relationships to GAL4 of Saccharomyces cerevisiae.

Abstract

Lactose or galactose induces the expression of the lactose-galactose regulon in Kluyveromyces lactis. We show here that the regulon is not induced in strains defective in L4C9. We demonstrate that this gene codes for a regulatory protein that acts in a positive manner to induce transcription. The LAC9 gene was isolated by complementation of a lac9 defective strain. DNA sequence analysis of the gene gave a deduced protein of 865 amino acids. Comparison of this sequence with that of the GAL4 protein of Saccharomyces cerevisiae revealed three regions of homology. One region of about 90 amino acid occurs at the amino terminus, which is known to mediate binding of GAL4 protein to upstream activator sequences. We speculate that a portion of this region, adjacent to the "metal-binding finger," specffies DNA binding. We discuss possible functions of the two other regions of homology. The functional implicatiodis of these structural similarities were examined. When LAC9 was introduced into a gal4 defective strain of S. cerevisiae it complemented the mutation and activated the galactose-melibiose regulon. However, LAC9 did not simply mimic GAL4. Unlike normal S. cerevisiae carrying GAL4, the strain carrying LAC9 gave constitutive expression of GAL) and MEL], two genes in the regulon. The strain did show glucose repression of the regulon, but repression was less severe with LAC9 than with GAL4. We discuss the implications of these results and how they may facilitate our understanding of the LAC9 and GAL4 regulatory proteins.Elucidation of genetic regulatory systems has relied heavily on the isolation of mutations in regulatory and structural genes. We are utilizing this approach to detetmine how lactose or galactose induces the lactose-galactose regulon in the yeast Kluyveromyces lactis. The regulon contains five known structural genes whose products are: LAC4, P-galactosidase (EC 3.2.1.23) (44); GAL], galactokinase (EC 2.7.1.6) (37); GAL7, galactose-1-phosphate uridyltransferase (EC 2.7.7.10) (37); GALIO, uridine diphosphoglucose-4-epimerase (EC 5.1.3.2) (37); and LAC12, a lactose permease (45). 3-Galactosidase activity can be induced over 100-fold above a moderate basal level (12). Increased enzyme activity results from increased transcription of the ,B-galactosidase structural gene, indicating that induction is regulated at the level of transcription (26). Mutations in LACIO cause constitutive expression of the regulon, suggesting that the gene functions in a negative fashion to regulate transcription (14).To further understand how lactose induces gene expression we characterized in more detail our previously isolated Lac-mutants (43). We found that mutants defective in lac9 are uninducible for all the enzymes of the lactose-galactose regulon. These and other data indicate that LAC9 is a positive regulatory gene that acts in trans to control transcription of target genes. This gene is the first positive-acting regulatory function that has been identified for the lactosegalactose regulon of K. lactis.We previously noted ...

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.

“…Previous data (17,19,28) showed that LAC9 and GAL4 bind the same family of UASs, and it had been argued that they have very similar DNA-binding domains (26). This hypothesis is supported also by the similarity of the hydroxyl radical footprints of LAC9 and GAL4 (Fig.…”

“…LAC9 enhances transcription of genes in the lactosegalactose regulon in the yeast Kluyveromyces lactis (28). 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.…”

Conserved functional domains of the RNA polymerase III general transcription factor BRF.

“…Strikingly, the BRF proteins of K. Iactis and C. albicans are 62% and 47% identical, respectively, to S. cerevisiae BRF. This conservation is much higher than that of gene-specific regulatory factors such as GAL4/ LAC9 and heat shock factor (HSF), where homologies between the K. lactis and S. cerevisiae proteins are confined to relatively short regions (Salmeron and Johnston 1986;Wray et al 1987;Jakobsen and Pelham 1991). The level of evolutionary conservation of BRF is similar to that of TFIIB, which is 67% identical between K. lactis and S. cerevisiae (Na and Hampsey 1993).…”

Telomere fusions caused by mutating the terminal region of telomeric DNA

“…All K. lactis cells used in this study are derivatives of the haploid 7B520 (16). Growth of yeast cells was carried out on standard media used for S. cerevisiae, either yeast extract͞peptone͞dextrose (YPD) or synthetic defined (SD) supplemented with appropriate nutrients.…”