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• W2056558725 abstract "Saccharomyces cerevisiae selectively uses good nitrogen sources (glutamine) in preference to poor ones (proline) by repressing GATA factor-dependent transcription of the genes needed to transport and catabolize poor nitrogen sources, a physiological process designated nitrogen catabolite repression (NCR). We show that some NCR-sensitive genes (CAN1,DAL5, DUR1,2, and DUR3) produce two transcripts of slightly different sizes. Synthesis of the shorter transcript is NCR-sensitive and that of the longer transcript is not. The longer transcript also predominates in gln3Δ mutants irrespective of the nitrogen source provided. We demonstrate that the longer mRNA species arises through the use of an alternative transcription start site generated by Gln3p-binding sites (GATAAs) being able to act as surrogate TATA elements. The ability of GATAAs to serve as surrogate TATAs, i.e. when synthesis of the shorter, NCR-sensitive transcripts are inhibited, correlates with sequestration of enhanced green fluorescent protein (EGFP)-Gln3p in the cytoplasm in a way that is indistinguishable from that seen with EGFP-Ure2p. However, when the shorter, NCR-sensitive DAL5transcript predominates, EGFP-Gln3p is nuclear. These data suggest that the mechanism underlying NCR involves the cytoplasmic association of Ure2p with Gln3p, an interaction that prevents Gln3p from reaching it is binding sites upstream of NCR-sensitive genes. Saccharomyces cerevisiae selectively uses good nitrogen sources (glutamine) in preference to poor ones (proline) by repressing GATA factor-dependent transcription of the genes needed to transport and catabolize poor nitrogen sources, a physiological process designated nitrogen catabolite repression (NCR). We show that some NCR-sensitive genes (CAN1,DAL5, DUR1,2, and DUR3) produce two transcripts of slightly different sizes. Synthesis of the shorter transcript is NCR-sensitive and that of the longer transcript is not. The longer transcript also predominates in gln3Δ mutants irrespective of the nitrogen source provided. We demonstrate that the longer mRNA species arises through the use of an alternative transcription start site generated by Gln3p-binding sites (GATAAs) being able to act as surrogate TATA elements. The ability of GATAAs to serve as surrogate TATAs, i.e. when synthesis of the shorter, NCR-sensitive transcripts are inhibited, correlates with sequestration of enhanced green fluorescent protein (EGFP)-Gln3p in the cytoplasm in a way that is indistinguishable from that seen with EGFP-Ure2p. However, when the shorter, NCR-sensitive DAL5transcript predominates, EGFP-Gln3p is nuclear. These data suggest that the mechanism underlying NCR involves the cytoplasmic association of Ure2p with Gln3p, an interaction that prevents Gln3p from reaching it is binding sites upstream of NCR-sensitive genes. nitrogen catabolite repression kilobase pair 4′,6-diamidino-2-phenylindole green fluorescent protein enhanced green fluorescent protein Arginine transport into Saccharomyces cerevisiae is mediated by several permeases; one is the low K m (10 μm) basic amino acid permease responsible for arginine uptake at low external concentrations. This “biosynthetic” permease is encoded byARGP/CAN1 (1.Grenson M. Mousset M. Wiame J.M. Bechet J. Biochim. Biophys. Acta. 1966; 127: 325-338Crossref PubMed Scopus (271) Google Scholar). Kinetic characterization of arginine transport by Can1p was conducted in ammonia-grown cells (1.Grenson M. Mousset M. Wiame J.M. Bechet J. Biochim. Biophys. Acta. 1966; 127: 325-338Crossref PubMed Scopus (271) Google Scholar). That Can1p is produced and functions in ammonia medium distinguished it from the high K m, “catabolic” general amino permease (GAP1) that transports a variety of amino acids (2.Grenson M. Hou C. Crabeel M. J. Bacteriol. 1970; 103: 770-777Crossref PubMed Google Scholar). Gap1p is produced only in medium containing a poor nitrogen source (2.Grenson M. Hou C. Crabeel M. J. Bacteriol. 1970; 103: 770-777Crossref PubMed Google Scholar, 3.Cooper T.G. Strathern J.N. Jones E.W. Broach J. The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 39-99Google Scholar). These early studies gave rise to the accepted notion thatCAN1 expression was insensitive to nitrogen catabolite repression (NCR).1NCR is the physiological process by which good nitrogen sources (glutamine, asparagine, and ammonia) are used in preference to poor ones (proline, allantoin, γ-aminobutyrate, and glutamate) (3.Cooper T.G. Strathern J.N. Jones E.W. Broach J. The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 39-99Google Scholar, 4.ter Schure E.G. van Riel N.A. Verrips C.T. FEMS Microbiol. Rev. 2000; 24: 67-83Crossref PubMed Scopus (205) Google Scholar, 5.Cooper T.G. Mycota. 1996; 3: 139-169Google Scholar). NCR-sensitive transcription of genes required for uptake and catabolism of poor nitrogen sources is mediated by UAS NTRelements (4.ter Schure E.G. van Riel N.A. Verrips C.T. FEMS Microbiol. Rev. 2000; 24: 67-83Crossref PubMed Scopus (205) Google Scholar, 5.Cooper T.G. Mycota. 1996; 3: 139-169Google Scholar). UAS NTR, also referred to as GATA elements since they contain the core sequence GATAA, are the binding sites for the NCR-sensitive transcriptional activators Gln3p and Gat1p/Nil1p (4.ter Schure E.G. van Riel N.A. Verrips C.T. FEMS Microbiol. Rev. 2000; 24: 67-83Crossref PubMed Scopus (205) Google Scholar, 5.Cooper T.G. Mycota. 1996; 3: 139-169Google Scholar). In the presence of excess nitrogen, Ure2p, a cytoplasmic prion-precursor protein (6.Wickner R.B. Science. 1994; 264: 566-569Crossref PubMed Scopus (1073) Google Scholar, 7.Maddelein M.-L. Wickner R.B. Mol. Cell Biol. 1999; 19: 4516-4524Crossref PubMed Scopus (86) Google Scholar), inhibits the operation of Gln3p and Gat1p (8.Courchesne W.E. Magasanik B. J. Bacteriol. 1988; 170: 708-713Crossref PubMed Scopus (129) Google Scholar, 9.Coffman J.A. Rai R. Cunningham T. Svetlov V. Cooper T.G. Mol. Cell. Biol. 1996; 16: 847-858Crossref PubMed Scopus (121) Google Scholar, 10.Coffman J.A. el Berry H.M. Cooper T.G. J. Bacteriol. 1994; 176: 7476-7483Crossref PubMed Google Scholar, 11.Drillen R. Aigle M. Lacroute F. Biochem. Biophys. Res. Commun. 1973; 53: 367-372Crossref PubMed Scopus (61) Google Scholar, 12.Drillien R. Lacroute F. J. Bacteriol. 1972; 109: 203-208Crossref PubMed Google Scholar). The observation that antibody against a fusion protein containing the Gln3p GATA-zinc finger could immunoprecipitate a Ure2p sized protein, when both proteins were overproduced, suggested that Ure2p regulation of Gln3p involved formation of a Gln3p-Ure2p complex (13.Blinder D. Coschigano P. Magasanik B. Bacteriol. 1996; 178: 4734-4736Crossref PubMed Google Scholar). A similar mechanism was hypothesized, although not demonstrated, for Ure2p regulation of Gat1p.A paradox concerning Can1p synthesis began to emerge in a study of the role played by Can1p in the NCR sensitivity of arginine-inducedCAR1 (arginase) expression (14.Cooper T.G. Kovari L. Sumrada R.A. Park H.-D. Luche R.M. Kovari I. J. Bacteriol. 1991; 174: 48-55Crossref Google Scholar). The critical observation was that heterologous overproduction of Can1p (driven by theADH1 promoter) resulted in arginine-induced CAR1expression becoming insensitive to NCR (14.Cooper T.G. Kovari L. Sumrada R.A. Park H.-D. Luche R.M. Kovari I. J. Bacteriol. 1991; 174: 48-55Crossref Google Scholar). This result suggested that a portion of NCR sensitivity of CAR1 expression was derived from NCR-sensitive inducer exclusion (14.Cooper T.G. Kovari L. Sumrada R.A. Park H.-D. Luche R.M. Kovari I. J. Bacteriol. 1991; 174: 48-55Crossref Google Scholar). NCR-sensitiveCAN1 expression was subsequently demonstrated by Northern blot analysis (15.Daugherty J.R. Rai R. ElBerry H.M. Cooper T.G. J. Bacteriol. 1993; 175: 64-73Crossref PubMed Google Scholar). The paradox arises in trying to rectify that the original characterization of Argp/Can1p was performed under conditions of strong NCR with the observation that CAN1 expression is highly NCR-sensitive.The present work provides a potential explanation of this paradox. Two mRNA species are transcribed from CAN1. One species is Gln3p-dependent and NCR-sensitive, and a second, slightly longer species, is present only under opposite conditions,i.e. strong NCR or deletion of GLN3. The longer mRNA is much less abundant than the shorter one. Similar profiles are also observed with DAL5, DUR1,2, andDUR3. We find that the CAN1 GATA sequences are available, in gln3Δ mutants and during growth with glutamine as nitrogen source, to serve as surrogate TATA elements. Utilization of an upstream start site accounts for the increased size of the NCR-insensitive transcript. Finally, EGFP-Gln3p is nuclear during times of Gln3p-dependent gene expression. When that expression is inhibited, EGRP-Gln3p is localized to the cytoplasm. This may account for the exposure of GATA sequences and their availability to serve as surrogate TATA elements when the function of Gln3p is inhibited or its cognate gene is deleted.DISCUSSIONWe have shown that CAN1 expression generates two transcripts as follows: (i) a rapidly migrating species with a profile characteristic of NCR-sensitive, Gln3p-dependent expression, and (ii) a less rapidly migrating species whose profile is just the opposite, present only when RNA is prepared from wild type cells cultured in glutamine medium or in gln3Δ andgln3Δgat1Δ mutants. The requirement of the clusteredCAN1 GATAA or TATAA sequences for the upper species to appear suggests that these GATA sequences can serve as surrogate TATAA elements under repressive growth conditions or when GLN3 is deleted. Similar functioning of GATAA sequences as surrogate TATA elements has been reported for higher eucaryotic cells (26.Singer V.L. Wobbe C.R. Struhl K. Genes Dev. 1990; 4: 636-645Crossref PubMed Scopus (154) Google Scholar, 27.Fong T.C. Emerson B.M. Genes Dev. 1992; 6: 521-532Crossref PubMed Scopus (69) Google Scholar, 28.Aird W.C. Parvin J.D. Sharp P.A. Rosenberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 269: 883-889Google Scholar). The presence of “variant TATAs” (GATAAs) situated upstream of the normalCAN1 TATAA can account for the production of the longerCAN1 mRNA species we observed earlier (15.Daugherty J.R. Rai R. ElBerry H.M. Cooper T.G. J. Bacteriol. 1993; 175: 64-73Crossref PubMed Google Scholar, 19.Coffman J.A. Rai R. Cooper T.G. J. Bacteriol. 1995; 177: 6910-6918Crossref PubMed Google Scholar). Our EGFP-Gln3p and EGFP-Ure2p experiments correlate the localization of Gln3p with its ability to support transcription. When Gln3p-mediated transcription is high, Gln3p is localized to the nucleus, and when transcription is minimal, Gln3p is cytoplasmic. The nearly identical intracellular localization of EGFP-Gln3p in a cell overproducing Ure2p and EGFP-Ure2p is consistent with the formation of a Ure2p-Gln3p complex as suggested earlier (13.Blinder D. Coschigano P. Magasanik B. Bacteriol. 1996; 178: 4734-4736Crossref PubMed Google Scholar). When these correlations are considered in light of experiments showing that the extent of NCR-sensitive transcription depends upon the relative levels of GATA factor and Ure2p production (39.Cunningham T.S. Andhare R. Cooper T.G. J. Biol. Chem. 2000; 275: 14408-14414Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), they argue that NCR is imposed by Gln3p complexing with Ure2p which prevents it from reaching its target GATAA-binding sites in the nucleus.During the preparation of this manuscript, four reports of experiments investigating the role played by TOR proteins in starvation appeared simultaneously and reached conclusions more or less similar to one another and to ours (30.Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (793) Google Scholar, 31.Cardenas M.E. Cutler N.S. Lorenz M. Di Como C.J. Heitman J. Genes Dev. 1999; 13: 3271-3279Crossref PubMed Scopus (473) Google Scholar, 32.Hardwick J.S. Kuruvilla F.G. Tong J.K. Shamji A.F. Schreiber S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14866-14870Crossref PubMed Scopus (464) Google Scholar, 33.Hardwick J.S. Tong J.K. Schreiber S.L. Nat. Genet. 1999; 23: 49Crossref Google Scholar). The most detailed experiments were those of Hall and collaborators (30.Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (793) Google Scholar) which led them to conclude that when cells grown in rich medium are treated with the immunosuppressant drug, rapamycin, Gln3p is dephosphorylated in a Sit4p-dependent manner and enters the nucleus. In rich medium, Gln3p is predominantly cytoplasmic. An earlier report by Edskes et al. (34.Edskes H.K. Hanover J.A. Wickner R.B. Genetics. 1999; 153: 585-594PubMed Google Scholar) identified Mks1p as another of the regulatory molecules that compose the signal transduction pathway that regulates Gln3p operation. Therefore, regulation of NCR-sensitive transcription can be anticipated to share characteristics of the cytoplasmic-nuclear trafficking that regulates operation of the PHO and stress-sensitive transcription factors (35.Oshima Y. Genes Genet. Syst. 1997; 72: 323-334Crossref PubMed Scopus (207) Google Scholar, 36.Gorner W. Durchschlag E. Martinez-Pastor M.T. Estruch F. Ammerer G. Hamilton B. Ruis H. Schuller C. Genes Dev. 1998; 12: 586-597Crossref PubMed Scopus (592) Google Scholar, 37.Reiser V. Ruis H. Ammerer G. Mol. Biol. Cell. 1999; 10: 1147-1161Crossref PubMed Scopus (176) Google Scholar). However, much remains to be done before all of the existing (and sometimes conflicting) data can be rectified, and a complete, cohesive picture of NCR-sensitive transcription and its relation to nitrogen starvation is understood. In this regard, it is pertinent to note that although some effects of nitrogen starvation are similar to those observed in relief of NCR, the two processes have been shown to be quite distinct (38.Beeser A.E. Cooper T.G. J. Bacteriol. 1999; 181: 2472-2476Crossref PubMed Google Scholar). Therefore, it would be surprising if the nitrogen starvation and NCR control pathways are found to be identical. Arginine transport into Saccharomyces cerevisiae is mediated by several permeases; one is the low K m (10 μm) basic amino acid permease responsible for arginine uptake at low external concentrations. This “biosynthetic” permease is encoded byARGP/CAN1 (1.Grenson M. Mousset M. Wiame J.M. Bechet J. Biochim. Biophys. Acta. 1966; 127: 325-338Crossref PubMed Scopus (271) Google Scholar). Kinetic characterization of arginine transport by Can1p was conducted in ammonia-grown cells (1.Grenson M. Mousset M. Wiame J.M. Bechet J. Biochim. Biophys. Acta. 1966; 127: 325-338Crossref PubMed Scopus (271) Google Scholar). That Can1p is produced and functions in ammonia medium distinguished it from the high K m, “catabolic” general amino permease (GAP1) that transports a variety of amino acids (2.Grenson M. Hou C. Crabeel M. J. Bacteriol. 1970; 103: 770-777Crossref PubMed Google Scholar). Gap1p is produced only in medium containing a poor nitrogen source (2.Grenson M. Hou C. Crabeel M. J. Bacteriol. 1970; 103: 770-777Crossref PubMed Google Scholar, 3.Cooper T.G. Strathern J.N. Jones E.W. Broach J. The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 39-99Google Scholar). These early studies gave rise to the accepted notion thatCAN1 expression was insensitive to nitrogen catabolite repression (NCR).1 NCR is the physiological process by which good nitrogen sources (glutamine, asparagine, and ammonia) are used in preference to poor ones (proline, allantoin, γ-aminobutyrate, and glutamate) (3.Cooper T.G. Strathern J.N. Jones E.W. Broach J. The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 39-99Google Scholar, 4.ter Schure E.G. van Riel N.A. Verrips C.T. FEMS Microbiol. Rev. 2000; 24: 67-83Crossref PubMed Scopus (205) Google Scholar, 5.Cooper T.G. Mycota. 1996; 3: 139-169Google Scholar). NCR-sensitive transcription of genes required for uptake and catabolism of poor nitrogen sources is mediated by UAS NTRelements (4.ter Schure E.G. van Riel N.A. Verrips C.T. FEMS Microbiol. Rev. 2000; 24: 67-83Crossref PubMed Scopus (205) Google Scholar, 5.Cooper T.G. Mycota. 1996; 3: 139-169Google Scholar). UAS NTR, also referred to as GATA elements since they contain the core sequence GATAA, are the binding sites for the NCR-sensitive transcriptional activators Gln3p and Gat1p/Nil1p (4.ter Schure E.G. van Riel N.A. Verrips C.T. FEMS Microbiol. Rev. 2000; 24: 67-83Crossref PubMed Scopus (205) Google Scholar, 5.Cooper T.G. Mycota. 1996; 3: 139-169Google Scholar). In the presence of excess nitrogen, Ure2p, a cytoplasmic prion-precursor protein (6.Wickner R.B. Science. 1994; 264: 566-569Crossref PubMed Scopus (1073) Google Scholar, 7.Maddelein M.-L. Wickner R.B. Mol. Cell Biol. 1999; 19: 4516-4524Crossref PubMed Scopus (86) Google Scholar), inhibits the operation of Gln3p and Gat1p (8.Courchesne W.E. Magasanik B. J. Bacteriol. 1988; 170: 708-713Crossref PubMed Scopus (129) Google Scholar, 9.Coffman J.A. Rai R. Cunningham T. Svetlov V. Cooper T.G. Mol. Cell. Biol. 1996; 16: 847-858Crossref PubMed Scopus (121) Google Scholar, 10.Coffman J.A. el Berry H.M. Cooper T.G. J. Bacteriol. 1994; 176: 7476-7483Crossref PubMed Google Scholar, 11.Drillen R. Aigle M. Lacroute F. Biochem. Biophys. Res. Commun. 1973; 53: 367-372Crossref PubMed Scopus (61) Google Scholar, 12.Drillien R. Lacroute F. J. Bacteriol. 1972; 109: 203-208Crossref PubMed Google Scholar). The observation that antibody against a fusion protein containing the Gln3p GATA-zinc finger could immunoprecipitate a Ure2p sized protein, when both proteins were overproduced, suggested that Ure2p regulation of Gln3p involved formation of a Gln3p-Ure2p complex (13.Blinder D. Coschigano P. Magasanik B. Bacteriol. 1996; 178: 4734-4736Crossref PubMed Google Scholar). A similar mechanism was hypothesized, although not demonstrated, for Ure2p regulation of Gat1p. A paradox concerning Can1p synthesis began to emerge in a study of the role played by Can1p in the NCR sensitivity of arginine-inducedCAR1 (arginase) expression (14.Cooper T.G. Kovari L. Sumrada R.A. Park H.-D. Luche R.M. Kovari I. J. Bacteriol. 1991; 174: 48-55Crossref Google Scholar). The critical observation was that heterologous overproduction of Can1p (driven by theADH1 promoter) resulted in arginine-induced CAR1expression becoming insensitive to NCR (14.Cooper T.G. Kovari L. Sumrada R.A. Park H.-D. Luche R.M. Kovari I. J. Bacteriol. 1991; 174: 48-55Crossref Google Scholar). This result suggested that a portion of NCR sensitivity of CAR1 expression was derived from NCR-sensitive inducer exclusion (14.Cooper T.G. Kovari L. Sumrada R.A. Park H.-D. Luche R.M. Kovari I. J. Bacteriol. 1991; 174: 48-55Crossref Google Scholar). NCR-sensitiveCAN1 expression was subsequently demonstrated by Northern blot analysis (15.Daugherty J.R. Rai R. ElBerry H.M. Cooper T.G. J. Bacteriol. 1993; 175: 64-73Crossref PubMed Google Scholar). The paradox arises in trying to rectify that the original characterization of Argp/Can1p was performed under conditions of strong NCR with the observation that CAN1 expression is highly NCR-sensitive. The present work provides a potential explanation of this paradox. Two mRNA species are transcribed from CAN1. One species is Gln3p-dependent and NCR-sensitive, and a second, slightly longer species, is present only under opposite conditions,i.e. strong NCR or deletion of GLN3. The longer mRNA is much less abundant than the shorter one. Similar profiles are also observed with DAL5, DUR1,2, andDUR3. We find that the CAN1 GATA sequences are available, in gln3Δ mutants and during growth with glutamine as nitrogen source, to serve as surrogate TATA elements. Utilization of an upstream start site accounts for the increased size of the NCR-insensitive transcript. Finally, EGFP-Gln3p is nuclear during times of Gln3p-dependent gene expression. When that expression is inhibited, EGRP-Gln3p is localized to the cytoplasm. This may account for the exposure of GATA sequences and their availability to serve as surrogate TATA elements when the function of Gln3p is inhibited or its cognate gene is deleted. DISCUSSIONWe have shown that CAN1 expression generates two transcripts as follows: (i) a rapidly migrating species with a profile characteristic of NCR-sensitive, Gln3p-dependent expression, and (ii) a less rapidly migrating species whose profile is just the opposite, present only when RNA is prepared from wild type cells cultured in glutamine medium or in gln3Δ andgln3Δgat1Δ mutants. The requirement of the clusteredCAN1 GATAA or TATAA sequences for the upper species to appear suggests that these GATA sequences can serve as surrogate TATAA elements under repressive growth conditions or when GLN3 is deleted. Similar functioning of GATAA sequences as surrogate TATA elements has been reported for higher eucaryotic cells (26.Singer V.L. Wobbe C.R. Struhl K. Genes Dev. 1990; 4: 636-645Crossref PubMed Scopus (154) Google Scholar, 27.Fong T.C. Emerson B.M. Genes Dev. 1992; 6: 521-532Crossref PubMed Scopus (69) Google Scholar, 28.Aird W.C. Parvin J.D. Sharp P.A. Rosenberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 269: 883-889Google Scholar). The presence of “variant TATAs” (GATAAs) situated upstream of the normalCAN1 TATAA can account for the production of the longerCAN1 mRNA species we observed earlier (15.Daugherty J.R. Rai R. ElBerry H.M. Cooper T.G. J. Bacteriol. 1993; 175: 64-73Crossref PubMed Google Scholar, 19.Coffman J.A. Rai R. Cooper T.G. J. Bacteriol. 1995; 177: 6910-6918Crossref PubMed Google Scholar). Our EGFP-Gln3p and EGFP-Ure2p experiments correlate the localization of Gln3p with its ability to support transcription. When Gln3p-mediated transcription is high, Gln3p is localized to the nucleus, and when transcription is minimal, Gln3p is cytoplasmic. The nearly identical intracellular localization of EGFP-Gln3p in a cell overproducing Ure2p and EGFP-Ure2p is consistent with the formation of a Ure2p-Gln3p complex as suggested earlier (13.Blinder D. Coschigano P. Magasanik B. Bacteriol. 1996; 178: 4734-4736Crossref PubMed Google Scholar). When these correlations are considered in light of experiments showing that the extent of NCR-sensitive transcription depends upon the relative levels of GATA factor and Ure2p production (39.Cunningham T.S. Andhare R. Cooper T.G. J. Biol. Chem. 2000; 275: 14408-14414Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), they argue that NCR is imposed by Gln3p complexing with Ure2p which prevents it from reaching its target GATAA-binding sites in the nucleus.During the preparation of this manuscript, four reports of experiments investigating the role played by TOR proteins in starvation appeared simultaneously and reached conclusions more or less similar to one another and to ours (30.Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (793) Google Scholar, 31.Cardenas M.E. Cutler N.S. Lorenz M. Di Como C.J. Heitman J. Genes Dev. 1999; 13: 3271-3279Crossref PubMed Scopus (473) Google Scholar, 32.Hardwick J.S. Kuruvilla F.G. Tong J.K. Shamji A.F. Schreiber S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14866-14870Crossref PubMed Scopus (464) Google Scholar, 33.Hardwick J.S. Tong J.K. Schreiber S.L. Nat. Genet. 1999; 23: 49Crossref Google Scholar). The most detailed experiments were those of Hall and collaborators (30.Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (793) Google Scholar) which led them to conclude that when cells grown in rich medium are treated with the immunosuppressant drug, rapamycin, Gln3p is dephosphorylated in a Sit4p-dependent manner and enters the nucleus. In rich medium, Gln3p is predominantly cytoplasmic. An earlier report by Edskes et al. (34.Edskes H.K. Hanover J.A. Wickner R.B. Genetics. 1999; 153: 585-594PubMed Google Scholar) identified Mks1p as another of the regulatory molecules that compose the signal transduction pathway that regulates Gln3p operation. Therefore, regulation of NCR-sensitive transcription can be anticipated to share characteristics of the cytoplasmic-nuclear trafficking that regulates operation of the PHO and stress-sensitive transcription factors (35.Oshima Y. Genes Genet. Syst. 1997; 72: 323-334Crossref PubMed Scopus (207) Google Scholar, 36.Gorner W. Durchschlag E. Martinez-Pastor M.T. Estruch F. Ammerer G. Hamilton B. Ruis H. Schuller C. Genes Dev. 1998; 12: 586-597Crossref PubMed Scopus (592) Google Scholar, 37.Reiser V. Ruis H. Ammerer G. Mol. Biol. Cell. 1999; 10: 1147-1161Crossref PubMed Scopus (176) Google Scholar). However, much remains to be done before all of the existing (and sometimes conflicting) data can be rectified, and a complete, cohesive picture of NCR-sensitive transcription and its relation to nitrogen starvation is understood. In this regard, it is pertinent to note that although some effects of nitrogen starvation are similar to those observed in relief of NCR, the two processes have been shown to be quite distinct (38.Beeser A.E. Cooper T.G. J. Bacteriol. 1999; 181: 2472-2476Crossref PubMed Google Scholar). Therefore, it would be surprising if the nitrogen starvation and NCR control pathways are found to be identical. We have shown that CAN1 expression generates two transcripts as follows: (i) a rapidly migrating species with a profile characteristic of NCR-sensitive, Gln3p-dependent expression, and (ii) a less rapidly migrating species whose profile is just the opposite, present only when RNA is prepared from wild type cells cultured in glutamine medium or in gln3Δ andgln3Δgat1Δ mutants. The requirement of the clusteredCAN1 GATAA or TATAA sequences for the upper species to appear suggests that these GATA sequences can serve as surrogate TATAA elements under repressive growth conditions or when GLN3 is deleted. Similar functioning of GATAA sequences as surrogate TATA elements has been reported for higher eucaryotic cells (26.Singer V.L. Wobbe C.R. Struhl K. Genes Dev. 1990; 4: 636-645Crossref PubMed Scopus (154) Google Scholar, 27.Fong T.C. Emerson B.M. Genes Dev. 1992; 6: 521-532Crossref PubMed Scopus (69) Google Scholar, 28.Aird W.C. Parvin J.D. Sharp P.A. Rosenberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 269: 883-889Google Scholar). The presence of “variant TATAs” (GATAAs) situated upstream of the normalCAN1 TATAA can account for the production of the longerCAN1 mRNA species we observed earlier (15.Daugherty J.R. Rai R. ElBerry H.M. Cooper T.G. J. Bacteriol. 1993; 175: 64-73Crossref PubMed Google Scholar, 19.Coffman J.A. Rai R. Cooper T.G. J. Bacteriol. 1995; 177: 6910-6918Crossref PubMed Google Scholar). Our EGFP-Gln3p and EGFP-Ure2p experiments correlate the localization of Gln3p with its ability to support transcription. When Gln3p-mediated transcription is high, Gln3p is localized to the nucleus, and when transcription is minimal, Gln3p is cytoplasmic. The nearly identical intracellular localization of EGFP-Gln3p in a cell overproducing Ure2p and EGFP-Ure2p is consistent with the formation of a Ure2p-Gln3p complex as suggested earlier (13.Blinder D. Coschigano P. Magasanik B. Bacteriol. 1996; 178: 4734-4736Crossref PubMed Google Scholar). When these correlations are considered in light of experiments showing that the extent of NCR-sensitive transcription depends upon the relative levels of GATA factor and Ure2p production (39.Cunningham T.S. Andhare R. Cooper T.G. J. Biol. Chem. 2000; 275: 14408-14414Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), they argue that NCR is imposed by Gln3p complexing with Ure2p which prevents it from reaching its target GATAA-binding sites in the nucleus. During the preparation of this manuscript, four reports of experiments investigating the role played by TOR proteins in starvation appeared simultaneously and reached conclusions more or less similar to one another and to ours (30.Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (793) Google Scholar, 31.Cardenas M.E. Cutler N.S. Lorenz M. Di Como C.J. Heitman J. Genes Dev. 1999; 13: 3271-3279Crossref PubMed Scopus (473) Google Scholar, 32.Hardwick J.S. Kuruvilla F.G. Tong J.K. Shamji A.F. Schreiber S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14866-14870Crossref PubMed Scopus (464) Google Scholar, 33.Hardwick J.S. Tong J.K. Schreiber S.L. Nat. Genet. 1999; 23: 49Crossref Google Scholar). The most detailed experiments were those of Hall and collaborators (30.Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (793) Google Scholar) which led them to conclude that when cells grown in rich medium are treated with the immunosuppressant drug, rapamycin, Gln3p is dephosphorylated in a Sit4p-dependent manner and enters the nucleus. In rich medium, Gln3p is predominantly cytoplasmic. An earlier report by Edskes et al. (34.Edskes H.K. Hanover J.A. Wickner R.B. Genetics. 1999; 153: 585-594PubMed Google Scholar) identified Mks1p as another of the regulatory molecules that compose the signal transduction pathway that regulates Gln3p operation. Therefore, regulation of NCR-sensitive transcription can be anticipated to share characteristics of the cytoplasmic-nuclear trafficking that regulates operation of the PHO and stress-sensitive transcription factors (35.Oshima Y. Genes Genet. Syst. 1997; 72: 323-334Crossref PubMed Scopus (207) Google Scholar, 36.Gorner W. Durchschlag E. Martinez-Pastor M.T. Estruch F. Ammerer G. Hamilton B. Ruis H. Schuller C. Genes Dev. 1998; 12: 586-597Crossref PubMed Scopus (592) Google Scholar, 37.Reiser V. Ruis H. Ammerer G. Mol. Biol. Cell. 1999; 10: 1147-1161Crossref PubMed Scopus (176) Google Scholar). However, much remains to be done before all of the existing (and sometimes conflicting) data can be rectified, and a complete, cohesive picture of NCR-sensitive transcription and its relation to nitrogen starvation is understood. In this regard, it is pertinent to note that although some effects of nitrogen starvation are similar to those observed in relief of NCR, the two processes have been shown to be quite distinct (38.Beeser A.E. Cooper T.G. J. Bacteriol. 1999; 181: 2472-2476Crossref PubMed Google Scholar). Therefore, it would be surprising if the nitrogen starvation and NCR control pathways are found to be identical. We thank Dr. A. T. Abul-Hamd forURE2 pAA19, the UT Yeast Group for suggested improvements to the manuscript, and Tim Higgins for preparing the figures." @default.
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