putA Gene Research Articles

Proline accumulation by mutation or disruption of the proline oxidase gene improves resistance to freezing and desiccation stresses in Saccharomyces cerevisiae

We examined the role of intracellular proline under freezing and desiccation stress conditions in Saccharomyces cerevisiae. When cultured in liquid minimal medium, the proline-nonutilizing mutant containing the put1 mutation (proline oxidase-deficient) produced more intracellular proline, and increased the cell survival rate as compared to the wild-type strain after freezing and desiccation. We also constructed two PUT1 gene disruptants. PUT1-disrupted mutants in minimal medium supplemented with external proline at 0.1% accumulated higher proline levels than those of the control strains (17–22-fold). These disruptants also had a 2–5-fold increase in cell viability compared to the control strains after freezing and desiccation stresses. These results indicate that proline has a stress-protective function in yeast.

Effect of Mutations in Genes PH085 and PH04 on Proline Utilization in Yeast Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae can utilize proline as a single nitrogen source. We have found that the strains with mutations in gene PH085, a structural gene encoding cyclin-dependent phosphoprotein kinase, cannot grow on the proline-containing media. The ability to utilize proline is restored in strains of the pho85pho4genotype. We suggest that phosphoprotein kinase Pho85p is involved in either phosphorylation of a highly specific proline permease, Put4p, or in signaling proline concentration. The Pho4p protein that activates transcription of the PH05 gene, a structural gene of acid phosphatase, seems to participate in the negative regulation of the PUT1 and PUT2 genes encoding enzymes of proline catabolism, proline oxidase and Δ-pyrroline-5-carboxylate dehydrogenase. Thus, regulation of phosphorus and nitrogen metabolism have common elements.

An Lrp-type transcriptional regulator from Agrobacterium tumefaciens condenses more than 100 nucleotides of DNA into globular nucleoprotein complexes

The PutR protein of Agrobacterium tumefaciens positively regulates expression of the putA gene in response to exogenous proline, resulting in the utilization of proline as a source of carbon and nitrogen. PutR activity required a region of DNA extending more than 106 nt upstream of the putA transcription start site. Purified PutR bound to this region with high degree of affinity and repressed expression of the putR promoter in vitro. PutR also activated the putA promoter in vitro in the presence of proline, though less strongly than in whole cells. PutR protected a DNA interval extending from nucleotides −30 to −140, but protected only one helical face over most of this interval, suggesting that it may bind only to this face of the DNA. The addition of proline caused a slight decrease in binding affinity and altered DNase I protection patterns along the entire length of the binding site. PutR-DNA complexes were found by atomic force microscopy to be globular rather than elongated. Although the DNA fragment in these complexes was 190 nm in length, the length of the visible DNA was only 150 nm, indicating that 40 nm of DNA (115 nt) must be condensed with protein. PutR caused a net bend of this binding site, and under some conditions, proline shifted the center of this bend by one helical turn.

Transcriptional regulation and locations of Agrobacterium tumefaciens genes required for complete catabolism of octopine.

By screening for octopine-inducible gene expression, we previously identified all the genes required for utilization of octopine as a source of carbon, nitrogen, and energy. They are (i) octopine oxidase, which converts octopine to arginine and pyruvate and is encoded by the ooxAB operon, (ii) arginase, which converts arginine to ornithine and urea and is encoded by arcA, (iii) ornithine cyclodeaminase, which converts ornithine to proline and ammonia and is encoded by the homologous arcB and ocd genes, and (iv) proline dehydrogenase, which converts proline to glutamate and is encoded by putA. Here we describe the regulation and localization of each of these genes. The ooxA-ooxB-ocd operon was previously shown to reside on the Ti plasmid and to be directly inducible by octopine. The arcAB operon is directly inducible by arginine, while it is induced by octopine only in strains that can convert octopine to arginine. Ornithine may also be a direct inducer of arcAB. putA is directly inducible by proline, while induction by octopine and by arginine (and probably by ornithine) requires their conversion to proline. Genetic studies indicate that arcAB and putA are localized on a conjugal genetic element. This element can be transferred to other Agrobacterium tumefaciens strains by a mechanism that does not require recA-dependent homologous recombination. Transfer of this genetic element from A. tumefaciens R10 requires at least one tra gene found on its Ti plasmid, indicating that this element is not self-transmissible but is mobilizable by the Ti plasmid. The DNA containing the arcAB and putA genes comigrates with a 243-kb linear molecular weight standard on field inversion electrophoretic gels.

The Rhizobium meliloti putA gene: its role in the establishment of the symbiotic interaction with alfalfa.

Little is known about the energy sources used by rhizobia during colonization, invasion and root nodule formation on leguminous plants. We have recently reported that an impaired proline metabolism in rhizobium meliloti leads to a reduced nodulation efficiency and competitiveness on alfalfa roots. In the present study we have characterized the R. meliloti proline dehydrogenase gene (putA) and addressed the question of its role in symbiosis. This rhizobial gene encodes a 1224-amino-acid-long polypeptide which is homologous to enteric bacteria, Rhodobacter capsulatus and Bradyrhizobium japonicum PutA proteins. Like the situation in these bacteria, sequence analysis identified the proline dehydrogenase (PDH) and pyrroline-5-carboxylate dehydrogenase (P5CDH) domains in the R. meliloti putA-encoded protein. Beta-galactosidase assays performed with free-living cells carrying a putA-lacZ transcriptional fusion revealed that R. meliloti putA gene expression is induced by proline, autoregulated by its encoded product, and independent of the general nitrogen regulatory system (Ntr). In addition, analysis of putA expression during the different steps of the symbiotic interaction with alfalfa showed that expression of this gene is turned on by the root exudates (RE), during root invasion and nodule formation, but not in differentiated nitrogen-fixing bacteroids. Furthermore, we show that the PutA- phenotype leads to a significant reduction of alfalfa root colonization by R. meliloti.

The putA gene of Agrobacterium tumefaciens is transcriptionally activated in response to proline by an Lrp-like protein and is not autoregulated.

The Agrobacterium tumefaciens putA gene, which encodes proline dehydrogenase, is transcriptionally induced by exogenous proline. In contrast to the putA genes of enteric bacteria, the A. tumefaciens putA gene is not regulated by the PutA protein, as the putA promoter remained strongly proline inducible in strains lacking PutA. A putA null mutation increased the expression of the putA promoter under a variety of conditions. However, this mutation is predicted to increase the cytoplasmic concentration of proline, and this alone probably accounts for its effects on putA expression. The putA promoter was also strongly induced by valine, and the putA genotype did not affect expression by this gratuitous inducer. An open reading frame (ORF) encoding an Lrp-like protein was found transcribed divergently from putA. Disruption of this ORF, designated putR, abolished induction of the putA promoter by proline or valine. In addition to activating putA, PutR also repressed its own transcription, and this autorepression was only slightly affected by exogenous proline. The transcription start sites for the putA and putR genes are separated by 64 nucleotides, suggesting that PutR could regulate both promoters by binding to a single operator.

Isolation, DNA sequence analysis, and mutagenesis of a proline dehydrogenase gene (putA) from Bradyrhizobium japonicum

We report here the cloning and sequencing of the gene for proline dehydrogenase (putA) of Bradyrhizobium japonicum. An open reading frame coding for 1,016 amino acids was identified. The B. japonicum gene codes for a bifunctional protein with proline dehydrogenase and pyrroline-5-carboxylate (P5C) dehydrogenase activities, as it does in Escherichia coli and Salmonella typhimurium. Comparison of the sequences of these proteins with other proline and P5C dehydrogenase sequences identified proline dehydrogenase and P5C dehydrogenase catalytic domains. Within the proline dehydrogenation domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Saccharomyces cerevisiae put1, and Drosophila melanogaster slgA. Within the P5C dehydrogenase domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Bacillus subtilis ipa76d, and S. cerevisiae put2. A consensus catalytic site for semialdehyde dehydrogenase was observed in the P5C dehydrogenase domain. This suggests that the substrate for this domain may be the open-chain gamma-glutamylsemialdehyde, not its cyclized form, P5C. Unlike the gene isolated from E. coli, S. typhimurium, and K. pneumoniae, the B. japonicum putA gene does not appear to be part of an operon with the proline porter gene (putP). Additionally, the B. japonicum gene lacks the putative C-terminal regulatory domain present in the E. coli and S. typhimurium genes. The gene was disrupted by insertion of antibiotic resistance gene cassettes, which were then recombined into the bacterial chromosome. Symbiotically active mutant strains that were devoid of putA activity were isolated. With this proline dehydrogenase clone, we will test the hypothesis that putA in symbiotic nitrogen-fixing B. japonicum bacteroids is transcriptionally regulated by drought and other stresses.

Expression of the putA gene encoding proline dehydrogenase from Rhodobacter capsulatus is independent of NtrC regulation but requires an Lrp-like activator protein

Four Rhodobacter capsulatus mutants unable to grow with proline as the sole nitrogen source were isolated by random Tn5 mutagenesis. The Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments. DNA sequence analysis of this region revealed three open reading frames designated selD, putR, and putA. The putA gene codes for a protein of 1,127 amino acid residues which is homologous to PutA of Salmonella typhimurium and Escherichia coli. The central part of R. capsulatus PutA showed homology to proline dehydrogenase of Saccharomyces cerevisiae (Put1) and Drosophila melanogaster (SlgA). The C-terminal part of PutA exhibited homology to Put2 (pyrroline-5-carboxylate dehydrogenase) of S. cerevisiae and to aldehyde dehydrogenases from different organisms. Therefore, it seems likely that in R. capsulatus, as in enteric bacteria, both enzymatic steps for proline degradation are catalyzed by a single polypeptide (PutA). The deduced amino acid sequence of PutR (154 amino acid residues) showed homology to the small regulatory proteins Lrp, BkdR, and AsnC. The putR gene, which is divergently transcribed from putA, is essential for proline utilization and codes for an activator of putA expression. The expression of putA was induced by proline and was not affected by ammonia or other amino acids. In addition, putA expression was autoregulated by PutA itself. Mutations in glnB, nifR1 (ntrC), and NifR4 (ntrA encoding sigma 54) had no influence on put gene expression. The open reading frame located downstream of R. capsulatus putR exhibited strong homology to the E. coli selD gene, which is involved in selenium metabolism. R. capsulatus selD mutants exhibited a Put+ phenotype, demonstrating that selD is required neither for viability nor for proline utilization.

Roles of URE2 and GLN3 in the Proline Utilization Pathway in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae can use alternative nitrogen sources such as arginine, urea, allantoin, gamma-aminobutyrate, or proline when preferred nitrogen sources like glutamine, asparagine, or ammonium ions are unavailable in the environment. Utilization of alternative nitrogen sources requires the relief of nitrogen repression and induction of specific permeases and enzymes. The products of the GLN3 and URE2 genes are required for the appropriate transcription of many genes in alternative nitrogen assimilatory pathways. GLN3 appears to activate their transcription when good nitrogen sources are unavailable, and URE2 appears to repress their transcription when alternative nitrogen sources are not needed. The participation of nitrogen repression and the regulators GLN3 and URE2 in the proline utilization pathway was evaluated in this study. Comparison of PUT gene expression in cells grown in repressing or derepressing nitrogen sources, in the absence of the inducer proline, indicated that both PUT1 and PUT2 are regulated by nitrogen repression, although the effect on PUT2 is comparatively small. Recessive mutations in URE2 elevated expression of the PUT1 and PUT2 genes 5- to 10-fold when cells were grown on a nitrogen-repressing medium. Although PUT3, the proline utilization pathway transcriptional activator, is absolutely required for growth on proline as the sole nitrogen source, a put3 ure2 strain had somewhat elevated PUT gene expression, suggesting an effect of the ure2 mutation in the absence of the PUT3 product. PUT1 and PUT2 gene expression did not require the GLN3 activator protein for expression under either repressing or derepressing conditions. Therefore, regulation of the PUT genes by URE2 does not require a functional GLN3 protein. The effect of the ure2 mutation on the PUT genes is not due to increased internal proline levels. URE2 repression appears to be limited to nitrogen assimilatory systems and does not affect genes involved in carbon, inositol, or phosphate metabolism or in mating-type control and sporulation.

Proline Dehydrogenase Activity of the Transcriptional Repressor PutA Is Required for Induction of the put Operon by Proline (∗)

The proline utilization (put) operon from Salmonella typhimurium consists of the putP gene, encoding a proline transporter, and the putA gene, encoding an enzyme with both proline dehydrogenase and 1-pyrroline-5-carboxylate dehydrogenase activities. In addition to these two enzymatic activities, the PutA protein is a transcriptional repressor that regulates the expression of putP and putA in response to the availability of proline. We report the isolation of super-repressor mutants of PutA that decrease expression from the putA promoter in the presence or absence of proline. None of the mutants exhibited increased affinity for the DNA in the put regulatory region in vitro. Although DNA binding by wild-type PutA was prevented by the addition of proline and an artificial electron acceptor, DNA binding by the two strongest super-repressors was not prevented under identical conditions. The proline dehydrogenase activity of the purified mutant proteins showed altered kinetic properties (increased Km(Pro), reduced Vmax, or a completely null phenotype). The observation that these mutations simultaneously affect induction by proline and proline dehydrogenase activity suggests that a single proline-binding site is involved in both proline dehydrogenase activity and induction of the expression of the put operon. Furthermore, the results indicate that the proline dehydrogenase activity of PutA is essential for induction of the put operon by proline.

Cloning, sequencing and analysis of a gene encoding Escherichia coli proline dehydrogenase

Using a genomic subtraction technique, we cloned a DNA sequence that is present in wild-type Escherichia coli strain CSH4 but is missing in a presumptive proline dehydrogenase deletion mutant RM2. Experimental evidence indicated that the cloned fragment codes for proline dehydrogenase (EC 1.5.99.8) since RM2 cells transformed with a plasmid containing this sequence was able to survive on minimal medium supplemented with proline as the sole nitrogen and carbon sources. The cloned DNA fragment has an open reading frame of 3942 bp and encodes a protein of 1313 amino acids with a calculated M r of 143 808. The deduced amino acid sequence of the E. colli proline dehydrogenase has an 84.9% homology to the previously reported Salmonella typhimurium putA gene but it is 111 amino acids longer at the C-terminal than the latter.

Regulatory circuit for responses of nitrogen catabolic gene expression to the GLN3 and DAL80 proteins and nitrogen catabolite repression in Saccharomyces cerevisiae.

We demonstrate that expression of the UGA1, CAN1, GAP1, PUT1, PUT2, PUT4, and DAL4 genes is sensitive to nitrogen catabolite repression. The expression of all these genes, with the exception of UGA1 and PUT2, also required a functional GLN3 protein. In addition, GLN3 protein was required for expression of the DAL1, DAL2, DAL7, GDH1, and GDH2 genes. The UGA1, CAN1, GAP1, and DAL4 genes markedly increased their expression when the DAL80 locus, encoding a negative regulatory element, was disrupted. Expression of the GDH1, PUT1, PUT2, and PUT4 genes also responded to DAL80 disruption, but much more modestly. Expression of GLN1 and GDH2 exhibited parallel responses to the provision of asparagine and glutamine as nitrogen sources but did not follow the regulatory responses noted above for the nitrogen catabolic genes such as DAL5. Steady-state mRNA levels of both genes did not significantly decrease when glutamine was provided as nitrogen source but were lowered by the provision of asparagine. They also did not respond to disruption of DAL80.

Human liver glutamic γ-semialdehyde dehydrogenase: Structural relationship to the yeast enzyme

The amino acid sequences of nine tryptic peptides (containing altogether 105 amino acids) from human liver glutamic γ-semialdehyde dehydrogenase (hitherto designated as ALDH4) were found to correspond, at 33–66% identity, to segments from the yeast 1-pyrroline-5-carboxylate (P5C) dehydrogenase encoded by the PUT2 gene.

Oxidation of L-thiazolidine-4-carboxylate by L-proline dehydrogenase in Escherichia coli.

L-Thiazolidine-4-carboxylate (T4C, gamma-thioproline) is a toxic analogue of L-proline. T4C can be oxidized by Escherichia coli to form N-formylcysteine, which is hydrolysed to yield formate and cysteine. To determine if L-proline dehydrogenase (EC 1.5.99.8) catalyses T4C degradation, membrane fractions from E. coli were tested for T4C and proline oxidation activity. The specific activity for T4C oxidation in membranes from bacteria grown with 10 mM-proline was similar to the specific activity for proline oxidation and about 100 times that in membranes from bacteria grown without proline. Both oxidation activities were inactivated at 45 degrees C at the same rate. Membranes from a strain with a deletion of the putA gene encoding L-proline dehydrogenase or a strain with a putA::Tn5 insertion mutation had no detectable activity with either substrate. Although T4C was a simple competitive inhibitor of proline oxidation, proline inhibited T4C oxidation in a way that gave competitive but sigmoidal kinetics. At low concentrations, T4C induced proline dehydrogenase synthesis. Cysteine auxotrophs containing the putA::Tn5 mutation could still use T4C as a cysteine source, and bacteria with this mutation consumed oxygen in the presence of T4C at half the control rate. These results indicate that T4C is a substrate and an inducer of L-proline dehydrogenase but suggest that E. coli also contains a second enzyme catalysing T4C degradation.

Isolation of Constitutive Mutations Affecting the Proline Utilization Pathway in Saccharomyces cerevisiae and Molecular Analysis of the PUT3 Transcriptional Activator

The enzymes of the proline utilization pathway (the products of the PUT1 and PUT2 genes) in Saccharomyces cerevisiae are coordinately regulated by proline and the PUT3 transcriptional activator. To learn more about the control of this pathway, constitutive mutations in PUT3 as well as in other regulators were sought. A scheme using a gene fusion between PUT1 (S. cerevisiae proline oxidase) and galK (Escherichia coli galactokinase) was developed to select directly for constitutive mutations affecting the PUT1 promoter. These mutations were secondarily screened for their effects in trans on the promoter of the PUT2 (delta 1-pyrroline-5-carboxylate dehydrogenase) gene by using a PUT2-lacZ (E. coli beta-galactosidase) gene fusion. Three different classes of mutations were isolated. The major class consisted of semidominant constitutive PUT3 mutations that caused PUT2-lacZ expression to vary from 2 to 22 times the uninduced level. A single dominant mutation in a new locus called PUT5 resulted in low-level constitutive expression of PUT2-lacZ; this mutation was epistatic to the recessive, noninducible put3-75 allele. Recessive constitutive mutations were isolated that had pleiotropic growth defects; it is possible that these mutations are not specific to the proline utilization pathway but may be in genes that control several pathways. Since the PUT3 gene appears to have a major role in the regulation of this pathway, a molecular analysis was undertaken. This gene was cloned by functional complementation of the put3-75 mutation. Strains carrying a complete deletion of this gene are viable, proline nonutilizing, and indistinguishable in phenotype from the original put3-75 allele. The PUT3 gene encodes a 2.8-kilobase-pair transcript that is not regulated by proline at the level of RNA accumulation. The presence of the gene on a high-copy-number plasmid did not alter the regulation of one of its target genes, PUT2-lacZ, suggesting that the PUT3 gene product is not limiting and that a titratable repressor is not involved in the regulation of this pathway.

Isolation of constitutive mutations affecting the proline utilization pathway in Saccharomyces cerevisiae and molecular analysis of the PUT3 transcriptional activator.

The enzymes of the proline utilization pathway (the products of the PUT1 and PUT2 genes) in Saccharomyces cerevisiae are coordinately regulated by proline and the PUT3 transcriptional activator. To learn more about the control of this pathway, constitutive mutations in PUT3 as well as in other regulators were sought. A scheme using a gene fusion between PUT1 (S. cerevisiae proline oxidase) and galK (Escherichia coli galactokinase) was developed to select directly for constitutive mutations affecting the PUT1 promoter. These mutations were secondarily screened for their effects in trans on the promoter of the PUT2 (delta 1-pyrroline-5-carboxylate dehydrogenase) gene by using a PUT2-lacZ (E. coli beta-galactosidase) gene fusion. Three different classes of mutations were isolated. The major class consisted of semidominant constitutive PUT3 mutations that caused PUT2-lacZ expression to vary from 2 to 22 times the uninduced level. A single dominant mutation in a new locus called PUT5 resulted in low-level constitutive expression of PUT2-lacZ; this mutation was epistatic to the recessive, noninducible put3-75 allele. Recessive constitutive mutations were isolated that had pleiotropic growth defects; it is possible that these mutations are not specific to the proline utilization pathway but may be in genes that control several pathways. Since the PUT3 gene appears to have a major role in the regulation of this pathway, a molecular analysis was undertaken. This gene was cloned by functional complementation of the put3-75 mutation. Strains carrying a complete deletion of this gene are viable, proline nonutilizing, and indistinguishable in phenotype from the original put3-75 allele. The PUT3 gene encodes a 2.8-kilobase-pair transcript that is not regulated by proline at the level of RNA accumulation. The presence of the gene on a high-copy-number plasmid did not alter the regulation of one of its target genes, PUT2-lacZ, suggesting that the PUT3 gene product is not limiting and that a titratable repressor is not involved in the regulation of this pathway.

The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences.

The PUT1 and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene. Two upstream activation sequences (UASs) in the PUT1 promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2. Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction.

The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences.

The PUT1 and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene. Two upstream activation sequences (UASs) in the PUT1 promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2. Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction.

A regulatory region responsible for proline-specific induction of the yeast PUT2 gene is adjacent to its TATA box.

Deletion analysis of the promoter of the PUT2 gene that functions in the proline utilization pathway of Saccharomyces cerevisiae identified a PUT2 upstream activation site (UAS). It is contained within a single 40-base-pair (bp) region located immediately upstream of the TATA box and is both necessary and sufficient for proline induction. When placed upstream of a CYC7-lacZ gene fusion, the 40-bp sequence conferred proline regulation on CYC7-lacZ. A 35-bp deletion within the PUT2 UAS in an otherwise intact PUT2 promoter resulted in noninducible expression of a PUT2-lacZ gene fusion. When a plasmid bearing this UAS-deleted promoter was placed in a strain carrying a constitutive mutation in the positive regulatory gene PUT3, expression of PUT2-lacZ was not constitutive but occurred at levels below those found under noninducing conditions. In heterologous as well as homologous gene fusions, the PUT2 UAS appeared to be responsible for uninduced as well as proline-induced levels of expression. Although located immediately adjacent to the PUT2 UAS, the TATA box did not appear to play a regulatory role, as indicated by the results of experiments in which it was replaced by the CYC7 TATA box. A 26-bp sequence containing this TATA box was critical to the expression of PUT2, since a deletion of this region completely abolished transcriptional activity of the gene under both inducing and noninducing conditions. Our results indicate that the PUT2 promoter has a comparatively simple structure, requiring UAS and TATA sequences as well as the PUT3 gene product (directly or indirectly) for its expression.

A regulatory region responsible for proline-specific induction of the yeast PUT2 gene is adjacent to its TATA box.

Deletion analysis of the promoter of the PUT2 gene that functions in the proline utilization pathway of Saccharomyces cerevisiae identified a PUT2 upstream activation site (UAS). It is contained within a single 40-base-pair (bp) region located immediately upstream of the TATA box and is both necessary and sufficient for proline induction. When placed upstream of a CYC7-lacZ gene fusion, the 40-bp sequence conferred proline regulation on CYC7-lacZ. A 35-bp deletion within the PUT2 UAS in an otherwise intact PUT2 promoter resulted in noninducible expression of a PUT2-lacZ gene fusion. When a plasmid bearing this UAS-deleted promoter was placed in a strain carrying a constitutive mutation in the positive regulatory gene PUT3, expression of PUT2-lacZ was not constitutive but occurred at levels below those found under noninducing conditions. In heterologous as well as homologous gene fusions, the PUT2 UAS appeared to be responsible for uninduced as well as proline-induced levels of expression. Although located immediately adjacent to the PUT2 UAS, the TATA box did not appear to play a regulatory role, as indicated by the results of experiments in which it was replaced by the CYC7 TATA box. A 26-bp sequence containing this TATA box was critical to the expression of PUT2, since a deletion of this region completely abolished transcriptional activity of the gene under both inducing and noninducing conditions. Our results indicate that the PUT2 promoter has a comparatively simple structure, requiring UAS and TATA sequences as well as the PUT3 gene product (directly or indirectly) for its expression.