• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. Nov 1993; 13(11): 6866–6875.
PMCID: PMC364749

Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

Abstract

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.3M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Ammerer G. Identification, purification, and cloning of a polypeptide (PRTF/GRM) that binds to mating-specific promoter elements in yeast. Genes Dev. 1990 Feb;4(2):299–312. [PubMed]
  • Bender A, Sprague GF., Jr MAT alpha 1 protein, a yeast transcription activator, binds synergistically with a second protein to a set of cell-type-specific genes. Cell. 1987 Aug 28;50(5):681–691. [PubMed]
  • Bruhn L, Hwang-Shum JJ, Sprague GF., Jr The N-terminal 96 residues of MCM1, a regulator of cell type-specific genes in Saccharomyces cerevisiae, are sufficient for DNA binding, transcription activation, and interaction with alpha 1. Mol Cell Biol. 1992 Aug;12(8):3563–3572. [PMC free article] [PubMed]
  • Christ C, Tye BK. Functional domains of the yeast transcription/replication factor MCM1. Genes Dev. 1991 May;5(5):751–763. [PubMed]
  • Dolan JW, Fields S. Cell-type-specific transcription in yeast. Biochim Biophys Acta. 1991 Feb 16;1088(2):155–169. [PubMed]
  • Dranginis AM. Binding of yeast a1 and alpha 2 as a heterodimer to the operator DNA of a haploid-specific gene. Nature. 1990 Oct 18;347(6294):682–685. [PubMed]
  • Elble R, Tye BK. Both activation and repression of a-mating-type-specific genes in yeast require transcription factor Mcm1. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10966–10970. [PMC free article] [PubMed]
  • Fields S, Chaleff DT, Sprague GF., Jr Yeast STE7, STE11, and STE12 genes are required for expression of cell-type-specific genes. Mol Cell Biol. 1988 Feb;8(2):551–556. [PMC free article] [PubMed]
  • Fields S, Herskowitz I. The yeast STE12 product is required for expression of two sets of cell-type specific genes. Cell. 1985 Oct;42(3):923–930. [PubMed]
  • Flessel MC, Brake AJ, Thorner J. The MF alpha 1 gene of Saccharomyces cerevisiae: genetic mapping and mutational analysis of promoter elements. Genetics. 1989 Feb;121(2):223–236. [PMC free article] [PubMed]
  • Goutte C, Johnson AD. a1 protein alters the DNA binding specificity of alpha 2 repressor. Cell. 1988 Mar 25;52(6):875–882. [PubMed]
  • Graham R, Gilman M. Distinct protein targets for signals acting at the c-fos serum response element. Science. 1991 Jan 11;251(4990):189–192. [PubMed]
  • Grayhack EJ. The yeast alpha 1 and MCM1 proteins bind a single strand of their duplex DNA recognition site. Mol Cell Biol. 1992 Aug;12(8):3573–3582. [PMC free article] [PubMed]
  • Hagen DC, Sprague GF., Jr Induction of the yeast alpha-specific STE3 gene by the peptide pheromone a-factor. J Mol Biol. 1984 Oct 5;178(4):835–852. [PubMed]
  • Hwang-Shum JJ, Hagen DC, Jarvis EE, Westby CA, Sprague GF., Jr Relative contributions of MCM1 and STE12 to transcriptional activation of a- and alpha-specific genes from Saccharomyces cerevisiae. Mol Gen Genet. 1991 Jun;227(2):197–204. [PubMed]
  • Inokuchi K, Nakayama A, Hishinuma F. Identification of sequence elements that confer cell-type-specific control of MF alpha 1 expression in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Sep;7(9):3185–3193. [PMC free article] [PubMed]
  • Jarvis EE, Clark KL, Sprague GF., Jr The yeast transcription activator PRTF, a homolog of the mammalian serum response factor, is encoded by the MCM1 gene. Genes Dev. 1989 Jul;3(7):936–945. [PubMed]
  • Jarvis EE, Hagen DC, Sprague GF., Jr Identification of a DNA segment that is necessary and sufficient for alpha-specific gene control in Saccharomyces cerevisiae: implications for regulation of alpha-specific and a-specific genes. Mol Cell Biol. 1988 Jan;8(1):309–320. [PMC free article] [PubMed]
  • Johnson AD, Herskowitz I. A repressor (MAT alpha 2 Product) and its operator control expression of a set of cell type specific genes in yeast. Cell. 1985 Aug;42(1):237–247. [PubMed]
  • Keleher CA, Goutte C, Johnson AD. The yeast cell-type-specific repressor alpha 2 acts cooperatively with a non-cell-type-specific protein. Cell. 1988 Jun 17;53(6):927–936. [PubMed]
  • Keleher CA, Passmore S, Johnson AD. Yeast repressor alpha 2 binds to its operator cooperatively with yeast protein Mcm1. Mol Cell Biol. 1989 Nov;9(11):5228–5230. [PMC free article] [PubMed]
  • Klopotowski T, Wiater A. Synergism of aminotriazole and phosphate on the inhibition of yeast imidazole glycerol phosphate dehydratase. Arch Biochem Biophys. 1965 Dec;112(3):562–566. [PubMed]
  • Kronstad JW, Holly JA, MacKay VL. A yeast operator overlaps an upstream activation site. Cell. 1987 Jul 31;50(3):369–377. [PubMed]
  • Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. [PubMed]
  • Lipke PN, Wojciechowicz D, Kurjan J. AG alpha 1 is the structural gene for the Saccharomyces cerevisiae alpha-agglutinin, a cell surface glycoprotein involved in cell-cell interactions during mating. Mol Cell Biol. 1989 Aug;9(8):3155–3165. [PMC free article] [PubMed]
  • McNamara PT, Bolshoy A, Trifonov EN, Harrington RE. Sequence-dependent kinks induced in curved DNA. J Biomol Struct Dyn. 1990 Dec;8(3):529–538. [PubMed]
  • Norman C, Runswick M, Pollock R, Treisman R. Isolation and properties of cDNA clones encoding SRF, a transcription factor that binds to the c-fos serum response element. Cell. 1988 Dec 23;55(6):989–1003. [PubMed]
  • Passmore S, Elble R, Tye BK. A protein involved in minichromosome maintenance in yeast binds a transcriptional enhancer conserved in eukaryotes. Genes Dev. 1989 Jul;3(7):921–935. [PubMed]
  • Primig M, Winkler H, Ammerer G. The DNA binding and oligomerization domain of MCM1 is sufficient for its interaction with other regulatory proteins. EMBO J. 1991 Dec;10(13):4209–4218. [PMC free article] [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Sauer RT, Smith DL, Johnson AD. Flexibility of the yeast alpha 2 repressor enables it to occupy the ends of its operator, leaving the center free. Genes Dev. 1988 Jul;2(7):807–816. [PubMed]
  • Schröter H, Mueller CG, Meese K, Nordheim A. Synergism in ternary complex formation between the dimeric glycoprotein p67SRF, polypeptide p62TCF and the c-fos serum response element. EMBO J. 1990 Apr;9(4):1123–1130. [PMC free article] [PubMed]
  • Sengupta P, Cochran BH. The PRE and PQ box are functionally distinct yeast pheromone response elements. Mol Cell Biol. 1990 Dec;10(12):6809–6812. [PMC free article] [PubMed]
  • Sengupta P, Cochran BH. MAT alpha 1 can mediate gene activation by a-mating factor. Genes Dev. 1991 Oct;5(10):1924–1934. [PubMed]
  • Shaw PE. Ternary complex formation over the c-fos serum response element: p62TCF exhibits dual component specificity with contacts to DNA and an extended structure in the DNA-binding domain of p67SRF. EMBO J. 1992 Aug;11(8):3011–3019. [PMC free article] [PubMed]
  • Shaw PE, Schröter H, Nordheim A. The ability of a ternary complex to form over the serum response element correlates with serum inducibility of the human c-fos promoter. Cell. 1989 Feb 24;56(4):563–572. [PubMed]
  • Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. [PubMed]
  • Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. [PMC free article] [PubMed]
  • Singh A, Chen EY, Lugovoy JM, Chang CN, Hitzeman RA, Seeburg PH. Saccharomyces cerevisiae contains two discrete genes coding for the alpha-factor pheromone. Nucleic Acids Res. 1983 Jun 25;11(12):4049–4063. [PMC free article] [PubMed]
  • Sprague GF., Jr Combinatorial associations of regulatory proteins and the control of cell type in yeast. Adv Genet. 1990;27:33–62. [PubMed]
  • Struhl K, Davis RW. Production of a functional eukaryotic enzyme in Escherichia coli: cloning and expression of the yeast structural gene for imidazole-glycerolphosphate dehydratase (his3). Proc Natl Acad Sci U S A. 1977 Dec;74(12):5255–5259. [PMC free article] [PubMed]
  • Tan S, Ammerer G, Richmond TJ. Interactions of purified transcription factors: binding of yeast MAT alpha 1 and PRTF to cell type-specific, upstream activating sequences. EMBO J. 1988 Dec 20;7(13):4255–4264. [PMC free article] [PubMed]
  • Tan S, Richmond TJ. DNA binding-induced conformational change of the yeast transcriptional activator PRTF. Cell. 1990 Jul 27;62(2):367–377. [PubMed]
  • Yuan YO, Stroke IL, Fields S. Coupling of cell identity to signal response in yeast: interaction between the alpha 1 and STE12 proteins. Genes Dev. 1993 Aug;7(8):1584–1597. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...