We used cells carrying plasmids causing the overproduction of Gln3p, Ure2p, or both of these proteins to elucidate the ability of Ure2p to prevent the activation of gene expression by Gln3p in cells growing in a glutamine-containing medium. Our results indicate that Ure2p probably does not interfere with the binding of the GATA factor Gln3p to GATAAG sites but acts directly on Gln3p to block its ability to activate transcription.In Saccharomyces cerevisiae, the expression of a number of genes whose products enable the organism to use a variety of compounds as sources of nitrogen is activated by the product of the GLN3 gene, Gln3p. This zinc finger protein exerts its effect by binding to GATAAG sequences located upstream of the regulated genes (1). The product of another gene, URE2, prevents the activation of the regulated genes by Gln3p in cells growing with glutamine as the source of nitrogen (5, 6).We have cloned the GLN3 and URE2 genes on plasmids, enabling us to overproduce the products of these genes (4, 7). We now present the results of experiments with cells that overproduce either one or both of the products of these genes. Our experiments suggest that Ure2p acts directly on Gln3p, converting it to a form that is unable to activate transcription.As previously reported, overproduction of Gln3p results in very slow growth, with a mass doubling time of more than 10 h in minimal media (7). We examined the effects of Ure2p on the growth inhibition exerted by Gln3p. To this end, we transformed strain PM38 (MAT␣ leu2-3,112 ura3-52) with plasmid pPM49 (2 m GAL10-GLN3 [7]). We also transformed this strain with plasmids carrying the URE2 gene fused to GAL10 as well as the LEU2 gene in order to create strains that overproduce both Gln3p and Ure2p. The high-copy-number plasmid pLE-8 was constructed by inserting a 1.7-kb StuI-SalI fragment of p8 containing the GAL10-URE2 fusion into the BamHI site of YEp13 (2, 3). The resulting plasmid has a LEU2 selectable marker and the GAL10 upstream activating sequence driving the URE2 gene. The single-copy centromere plasmid pLC-8 (YCpGAL10-URE2) was constructed by replacing the URA3 fragment of the p8 plasmid with the SalIXhoI LEU2-containing fragment of YEp13.The results presented in Fig. 1 show that overproduction of Gln3p in the strain carrying the GLN3 gene fused to GAL10 causes strong growth inhibition and that growth is restored by the overproduction of Ure2p resulting from the simultaneous presence of the plasmid carrying URE2 fused to GAL10.The effect of Ure2p is also demonstrated by the ability of Ure2p to depress the ability of overproduced Gln3p to activate the synthesis of glutamine synthetase. As shown in Table 1, overproduction of Gln3p enables the cell to produce glutamine synthetase during growth with glutamine as the source of nitrogen, but this increase in the level of the enzyme is largely prevented by the simultaneous overproduction of Ure2p.We have previously shown that Ure2p blocks neither the FIG. 1. Suppression of GLN3 lethality by overexpression of URE2...

No abstract

We describe the purification of the product of the GLN3 gene of Saccharomyces cerevisiae and the demonstration that the purified product, Gln3p, binds specifically to the DNA sequences GATAAG and GATTAG, previously identified as nitrogen-responsive upstream activation sequences (UAS N ). When Gln3p is overproduced, it is released from the cells in a highly aggregated form incapable of specific binding to UAS N . We used Gln3p tagged with six histidine codons at the 5 terminus and equipped with a galactose-inducible promoter to overproduce histidine-tagged Gln3p. The material was denatured, adsorbed to an Ni-nitrilotriacetic acid (NTA)-agarose column, eluted with imidazole, and after renaturation further purified on a gel filtration column. We then demonstrated the specific binding of the more than 90% pure Gln3p to the UAS N by gel shift and footprinting methods.

The STES gene encodes an essential element of the pheromone response pathway which is known to act either after the G subunit encoded by the STE4 gene or at the same step. Mutations in STES, designated STESHYP, that partially activate the pathway in the absence of pheromone were isolated. One allele (STE5HrYP2) indicating that the STES product acts after the receptor (STE2 product) and after the G protein ,B and 'y subunits (STE4 and STE18 products, respectively). However, the phenotypes of the STESHYP mutations were less pronounced in ste4 and stel8 mutants, suggesting that the STE5HYP generated signal partially depends on the proposed G., complex. The STESHYP alleles did not suppress ste7, stell, stel2, orfi*s3 kssl null mutants, consistent with previous findings that the STES product acts before the protein kinases encoded by STE7, STE]], FUS3, and KSSI and the transcription factor encoded by STE12. The mating defects of the ste2 deletion mutant and the temperature-sensitive ste4-3 mutant were also suppressed by overexpression of wild-type STE5.The slow-growth phenotype manifested by cells carrying STESHYP alleles was enhanced by the sst2-1 mutation; this effect was eliminated in ste4 mutants. These results provide the first evidence that the STES gene product performs its function after the G protein subunits.The pheromone response in the yeast Saccharomyces cerevisiae provides a model system for studying general features of peptide-hormone action (see references 59 and 75 for reviews). In this yeast, conjugation of haploid a and a cells yields a/a diploid cells. Conjugation requires the action of peptide pheromones: a cells secrete a-factor pheromone and respond to a-factor, whereas a cells secrete a-factor pheromone and respond to a-factor. The binding of the pheromones to specific receptors on the target cell initiates the mating program. Specific cellular responses to pheromones include the arrest of cell division in G1 (34) and the production of cellular factors required for cell aggregation, cell fusion, karyogamy, and pheromone desensitization (17,18,54,60,72,79). These responses are mediated, at least in part, by changes in the transcription of cellular genes. Pheromones also provide spatial information which allows cells to locate and choose specific mating partners (42,43,51).Previous genetic analyses of the pheromone response pathway identified a number of components required for signal transduction. The STE2 and STE3 genes encode the a-factor and a-factor receptors, respectively (4, 35, 46, 57,

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32P04 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.We have previously demonstrated that the mRNA population in vegetatively growing amoebae of the cellular slime mold Dictyostelium discoideum decays with complex kinetics, consisting of at least two major components: a rapidly decaying component with a half-life of approximately 50 min, and a long-lived component with a half-life of approximately 10 h (14, 58). Similar complex kinetics have been observed for the decay of poly(A)+ RNA in a variety of other eucaryotic cells (5,25,39,57,77,78,81). It is likely that these complex decay kinetics reflect the sum of the decay rates of individual mRNAs (e.g., in D. discoideum, the most stable mRNAs have half-lives of approximately 10 h and the least stable mRNAs have half-lives which are approximately 10-fold shorter). It has been s...