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Mol Cell Biol. 1988 July; 8(7): 2690–2697.
PMCID: PMC363479

LEU3 of Saccharomyces cerevisiae activates multiple genes for branched-chain amino acid biosynthesis by binding to a common decanucleotide core sequence.

Abstract

LEU3 of Saccharomyces cerevisiae encodes an 886-amino-acid polypeptide that regulates transcription of a group of genes involved in leucine biosynthesis and has been shown to bind specifically to a 114-base-pair DNA fragment of the LEU2 upstream region (P. Friden and P. Schimmel, Mol. Cell. Biol. 7:2707-2717, 1987). We show here that, in addition to LEU2, LEU3 binds in vitro to sequences in the promoter regions of LEU1, LEU4, ILV2, and, by inference, ILV5. The largely conserved decanucleotide core sequence shared by the binding sites in these genes is CCGGNNCCGG. Methylation interference footprinting experiments show that LEU3 makes symmetrical contacts with the conserved bases that lie in the major groove. Synthetic oligonucleotides (19 to 29 base pairs) which contain the core decanucleotide and flanking sequences of LEU1, LEU2, LEU4, and ILV2 have individually been placed upstream of a LEU3-insensitive test promoter. The expression of each construction is activated by LEU3, although the degree of activation varies considerably according to the specific oligonucleotide which is introduced. A promoter construction with substitutions in the core sequence remains LEU3 insensitive, however. One of the oligonucleotides (based on a LEU2 sequence) was also tested and shown to confer leucine-sensitive expression on the test promoter. The results demonstrate that only a short sequence element is necessary for LEU3-dependent promoter binding and activation and provide direct evidence for an expanded repertoire of genes that are activated by LEU3.

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Selected References

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  • Andreadis A, Hsu YP, Hermodson M, Kohlhaw G, Schimmel P. Yeast LEU2. Repression of mRNA levels by leucine and primary structure of the gene product. J Biol Chem. 1984 Jul 10;259(13):8059–8062. [PubMed]
  • Armaleo D, Fischer M, Gross SR. Effect of alpha-isopropylmalate on the synthesis of RNA and protein in Neurospora. Mol Gen Genet. 1985;200(2):346–349. [PubMed]
  • Arndt K, Fink GR. GCN4 protein, a positive transcription factor in yeast, binds general control promoters at all 5' TGACTC 3' sequences. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8516–8520. [PubMed]
  • Friden P, Schimmel P. LEU3 of Saccharomyces cerevisiae encodes a factor for control of RNA levels of a group of leucine-specific genes. Mol Cell Biol. 1987 Aug;7(8):2708–2717. [PMC free article] [PubMed]
  • Fried M, Crothers DM. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. [PMC free article] [PubMed]
  • Garner MM, Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. [PMC free article] [PubMed]
  • Giniger E, Varnum SM, Ptashne M. Specific DNA binding of GAL4, a positive regulatory protein of yeast. Cell. 1985 Apr;40(4):767–774. [PubMed]
  • Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 1983;101:181–191. [PubMed]
  • Guarente L. Yeast promoters: positive and negative elements. Cell. 1984 Apr;36(4):799–800. [PubMed]
  • Guarente L, Mason T. Heme regulates transcription of the CYC1 gene of S. cerevisiae via an upstream activation site. Cell. 1983 Apr;32(4):1279–1286. [PubMed]
  • Hope IA, Struhl K. GCN4 protein, synthesized in vitro, binds HIS3 regulatory sequences: implications for general control of amino acid biosynthetic genes in yeast. Cell. 1985 Nov;43(1):177–188. [PubMed]
  • Hope IA, Struhl K. GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA. EMBO J. 1987 Sep;6(9):2781–2784. [PubMed]
  • Hsu YP, Kohlhaw GB, Niederberger P. Evidence that alpha-isopropylmalate synthase of Saccharomyces cerevisiae is under the "general" control of amino acid biosynthesis. J Bacteriol. 1982 May;150(2):969–972. [PMC free article] [PubMed]
  • Hsu YP, Schimmel P. Yeast LEU1. Repression of mRNA levels by leucine and relationship of 5'-noncoding region to that of LEU2. J Biol Chem. 1984 Mar 25;259(6):3714–3719. [PubMed]
  • Laughon A, Gesteland RF. Primary structure of the Saccharomyces cerevisiae GAL4 gene. Mol Cell Biol. 1984 Feb;4(2):260–267. [PMC free article] [PubMed]
  • Maniatis T, Goodbourn S, Fischer JA. Regulation of inducible and tissue-specific gene expression. Science. 1987 Jun 5;236(4806):1237–1245. [PubMed]
  • McKnight S, Tjian R. Transcriptional selectivity of viral genes in mammalian cells. Cell. 1986 Sep 12;46(6):795–805. [PubMed]
  • Messenguy F, Dubois E, Descamps F. Nucleotide sequence of the ARGRII regulatory gene and amino acid sequence homologies between ARGRII PPRI and GAL4 regulatory proteins. Eur J Biochem. 1986 May 15;157(1):77–81. [PubMed]
  • Miller J, McLachlan AD, Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985 Jun;4(6):1609–1614. [PubMed]
  • Olshan AR, Gross SR. Role of the leu-3 cistron in the regulation of the synthesis of isoleucine and valine biosynthetic enzymes of Neurospora. J Bacteriol. 1974 May;118(2):374–384. [PMC free article] [PubMed]
  • Pabo CO, Lewis M. The operator-binding domain of lambda repressor: structure and DNA recognition. Nature. 1982 Jul 29;298(5873):443–447. [PubMed]
  • Pfeifer K, Arcangioli B, Guarente L. Yeast HAP1 activator competes with the factor RC2 for binding to the upstream activation site UAS1 of the CYC1 gene. Cell. 1987 Apr 10;49(1):9–18. [PubMed]
  • Pfeifer K, Prezant T, Guarente L. Yeast HAP1 activator binds to two upstream activation sites of different sequence. Cell. 1987 Apr 10;49(1):19–27. [PubMed]
  • Polacco JC, Gross SR. The product of the leu-3 cistron as a regulatory element for the production of the leucine biosynthetic enzymes of Neurospora. Genetics. 1973 Jul;74(3):443–459. [PubMed]
  • Rothstein RJ. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. [PubMed]
  • Siebenlist U, Gilbert W. Contacts between Escherichia coli RNA polymerase and an early promoter of phage T7. Proc Natl Acad Sci U S A. 1980 Jan;77(1):122–126. [PubMed]
  • Struhl K. Promoters, activator proteins, and the mechanism of transcriptional initiation in yeast. Cell. 1987 May 8;49(3):295–297. [PubMed]
  • Takeda Y, Ohlendorf DH, Anderson WF, Matthews BW. DNA-binding proteins. Science. 1983 Sep 9;221(4615):1020–1026. [PubMed]
  • Thomas JH, Neff NF, Botstein D. Isolation and characterization of mutations in the beta-tubulin gene of Saccharomyces cerevisiae. Genetics. 1985 Dec;111(4):715–734. [PubMed]
  • Umbarger HE. Amino acid biosynthesis and its regulation. Annu Rev Biochem. 1978;47:532–606. [PubMed]
  • von Hippel PH, Bear DG, Morgan WD, McSwiggen JA. Protein-nucleic acid interactions in transcription: a molecular analysis. Annu Rev Biochem. 1984;53:389–446. [PubMed]
  • Zhou K, Brisco PR, Hinkkanen AE, Kohlhaw GB. Structure of yeast regulatory gene LEU3 and evidence that LEU3 itself is under general amino acid control. Nucleic Acids Res. 1987 Jul 10;15(13):5261–5273. [PMC free article] [PubMed]

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