Regulation of yeast LEU2. Total deletion of regulatory gene LEU3 unmasks GCN4-dependent basal level expression of LEU2.
We have constructed a total deletion of the regulatory gene LEU3. Comparing the deletion mutant with a leu3 spontaneous mutant, we find that both types of mutants have lost the ability to regulate a LEU2'-lacZ translational fusion by the LEU3-alpha-isopropylmalate-dependent mechanism, which we confirm to be the major regulatory mechanism for LEU2. Surprisingly, cells containing the total leu3 deletion are more leaky (i.e. grow better in the absence of extraneous leucine) than cells containing a spontaneous leu3 mutation. Accompanying the growth rate difference is a difference in the expression of the LEU2-lacZ fusion: the specific activity of beta-galactosidase amounts to about 8% of a wild type control in a leu3 total deletion mutant, but drops to about 2% in a leu3 spontaneous mutant. The spontaneous mutant differs from the total deletion mutant in that it produces an inactive protein which is still able to bind to the LEU2 upstream activating sequence. We conclude that a basal level control of LEU2 becomes manifest in the absence of LEU3 and is interfered with when LEU3 protein binds to the LEU2 promoter. This conclusion is supported by the finding that a mutant which contains an intact LEU3 gene but is unable to generate alpha-isopropylmalate also interferes with basal level expression of LEU2. Basal level expression depends upon the GCN4 protein, even though LEU2 is not subject to derepression by the general amino acid control system. Changes in the steady-state concentration of LEU2 mRNA show the same trend as changes in the specific activity of the LEU2-lacZ fusion protein, suggesting that regulation of LEU2 expression at both the basal and nonbasal levels is largely transcriptional. The role of alpha-isopropylmalate in the regulation of LEU2 expression appears to be that of a co-activator. Employing mobility shift assays, we show that specific interaction between the LEU3 protein and a 30-base pair DNA fragment carrying the upstream activating sequence of LEU2 takes place irrespective of the presence or absence of alpha-isopropylmalate.
Brisco
P R
PR
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Kohlhaw
G B
GB
D015079:2-Isopropylmalate Synthase / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D002872:Chromosome Deletion; D004926:Escherichia coli / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005809:Genes, Regulator; D007930:Leucine / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D010641:Phenotype; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development
D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; D015079:2-Isopropylmalate Synthase; D007930:Leucine