scholarly journals The Gal3p-Gal80p-Gal4p Transcription Switch of Yeast: Gal3p Destabilizes the Gal80p-Gal4p Complex in Response to Galactose and ATP

1999 ◽  
Vol 19 (11) ◽  
pp. 7828-7840 ◽  
Author(s):  
Alok Kumar Sil ◽  
Samina Alam ◽  
Ping Xin ◽  
Ly Ma ◽  
Melissa Morgan ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Genetic Interaction ◽  
Transcription Activation ◽  
Full Length ◽  
Signal Transducer ◽  
Binding Peptide ◽  
Two Hybrid

ABSTRACT The Gal3, Gal80, and Gal4 proteins of Saccharomyces cerevisiae comprise a signal transducer that governs the galactose-inducible Gal4p-mediated transcription activation ofGAL regulon genes. In the absence of galactose, Gal80p binds to Gal4p and prohibits Gal4p from activating transcription, whereas in the presence of galactose, Gal3p binds to Gal80p and relieves its inhibition of Gal4p. We have found that immunoprecipitation of full-length Gal4p from yeast extracts coprecipitates less Gal80p in the presence than in the absence of Gal3p, galactose, and ATP. We have also found that retention of Gal80p by GSTG4AD (amino acids [aa] 768 to 881) is markedly reduced in the presence compared to the absence of Gal3p, galactose, and ATP. Consistent with these in vitro results, an in vivo two-hybrid genetic interaction between Gal80p and Gal4p (aa 768 to 881) was shown to be weaker in the presence than in the absence of Gal3p and galactose. These compiled results indicate that the binding of Gal3p to Gal80p results in destabilization of a Gal80p-Gal4p complex. The destabilization was markedly higher for complexes consisting of G4AD (aa 768 to 881) than for full-length Gal4p, suggesting that Gal80p relocated to a second site on full-length Gal4p. Congruent with the idea of a second site, we discovered a two-hybrid genetic interaction involving Gal80p and the region of Gal4p encompassing aa 225 to 797, a region of Gal4p linearly remote from the previously recognized Gal80p binding peptide within Gal4p aa 768 to 881.

scholarly journals 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.

1992 ◽  
Vol 12 (8) ◽  
pp. 3563-3572 ◽  
Author(s):  
L Bruhn ◽  
J J Hwang-Shum ◽  
G F Sprague
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Dna Binding ◽  
Transcription Activation ◽  
Reporter Genes ◽  
Cell Type ◽  
Cell Type Specific

MCM1 performs several functions necessary for its role in regulating cell type-specific gene expression in the yeast Saccharomyces cerevisiae: DNA binding, transcription activation, and interaction with coregulatory proteins such as alpha 1. We analyzed a set of MCM1 deletion derivatives using in vivo reporter gene assays and in vitro DNA-binding studies to determine which regions of MCM1 are important for its various activities. We also analyzed a set of LexA-MCM1 hybrids to examine the ability of different segments of MCM1 to activate transcription independent of MCM1's DNA-binding function. The first third of MCM1 [MCM1(1-96)], which includes an 80-residue segment homologous to the mammalian serum response factor, was sufficient for high-affinity DNA binding, for activation of reporter gene expression, and for interaction with alpha 1 in vitro and in vivo. However, the ability of MCM1(1-96) to activate transcription and to interact with alpha 1 was somewhat reduced compared with wild-type MCM1 [MCM1(1-286)]. Optimal interaction with alpha 1 required residues 99 to 117, in which 18 of 19 amino acids are acidic in character. Optimal transcription activation required a segment from residues 188 to 286, in which 50% of the amino acids are glutamine. Deletion of this segment from MCM1 reduced expression of reporter genes by about twofold. Moreover, LexA-MCM1 hybrids containing this segment were able to activate expression of reporter genes that rely on LexA binding sites as potential upstream activation sequences. Thus, glutamine-rich regions may contribute to the activation function of yeast transcription activators, as has been suggested for glutamine-rich mammalian proteins such as Sp1.

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

1992 ◽  
Vol 12 (8) ◽  
pp. 3563-3572
Author(s):  
L Bruhn ◽  
J J Hwang-Shum ◽  
G F Sprague
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Dna Binding ◽  
Transcription Activation ◽  
Reporter Genes ◽  
Cell Type ◽  
Cell Type Specific

MCM1 performs several functions necessary for its role in regulating cell type-specific gene expression in the yeast Saccharomyces cerevisiae: DNA binding, transcription activation, and interaction with coregulatory proteins such as alpha 1. We analyzed a set of MCM1 deletion derivatives using in vivo reporter gene assays and in vitro DNA-binding studies to determine which regions of MCM1 are important for its various activities. We also analyzed a set of LexA-MCM1 hybrids to examine the ability of different segments of MCM1 to activate transcription independent of MCM1's DNA-binding function. The first third of MCM1 [MCM1(1-96)], which includes an 80-residue segment homologous to the mammalian serum response factor, was sufficient for high-affinity DNA binding, for activation of reporter gene expression, and for interaction with alpha 1 in vitro and in vivo. However, the ability of MCM1(1-96) to activate transcription and to interact with alpha 1 was somewhat reduced compared with wild-type MCM1 [MCM1(1-286)]. Optimal interaction with alpha 1 required residues 99 to 117, in which 18 of 19 amino acids are acidic in character. Optimal transcription activation required a segment from residues 188 to 286, in which 50% of the amino acids are glutamine. Deletion of this segment from MCM1 reduced expression of reporter genes by about twofold. Moreover, LexA-MCM1 hybrids containing this segment were able to activate expression of reporter genes that rely on LexA binding sites as potential upstream activation sequences. Thus, glutamine-rich regions may contribute to the activation function of yeast transcription activators, as has been suggested for glutamine-rich mammalian proteins such as Sp1.

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

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875 ◽  
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dna Sequences ◽  
Point Mutations ◽  
Transcription Activation ◽  
Specific Gene ◽  
Binding Assays ◽  
Weak Binding

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.

scholarly journals Definition of a minimal munc18c domain that interacts with syntaxin 4

2000 ◽  
Vol 350 (3) ◽  
pp. 741-746 ◽  
Author(s):  
Julian GRUSOVIN ◽  
Violet STOICHEVSKA ◽  
Keith H. GOUGH ◽  
Katrina NUNAN ◽  
Colin W. WARD ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Full Length ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Interaction Studies ◽  
Syntaxin 4 ◽  
Definition Of ◽  
Two Hybrid

munc18c is a critical protein involved in trafficking events associated with syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its interaction with syntaxin 4, fragments of munc18c were generated and their interaction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. In vitro protein–protein interaction studies were then used to confirm that the interaction between the proteins was direct. Full-length munc18c1–592, munc18c1–139 and munc18c1–225, but not munc18c226–592, munc18c1–100, munc18c43–139 or munc18c66–139, interacted with the cytoplasmic portion of syntaxin 4, Stx42–273, as assessed by yeast two-hybrid assay of growth on nutritionally deficient media and by β-galactosidase reporter induction. The N-terminal predicted helix-a-helix-b-helix-c region of syntaxin 4, Stx429–157, failed to interact with full-length munc18c1–592, indicating that a larger portion of syntaxin 4 is necessary for the interaction. The yeast two-hybrid results were confirmed by protein–protein interaction studies between Stx42–273 and glutathione S-transferase fusion proteins of munc18c. Full-length munc18c1–592, munc18c1–139 and munc18c1–225 interacted with Stx42–273 whereas munc18c1–100 did not, consistent with the yeast two-hybrid data. These data thus identify a region of munc18c between residues 1 and 139 as a minimal domain for its interaction with syntaxin 4.

scholarly journals Modular structure of chromosomal proteins HMG-14 and HMG-17: definition of a transcriptional enhancement domain distinct from the nucleosomal binding domain.

1995 ◽  
Vol 15 (12) ◽  
pp. 6663-6669 ◽  
Author(s):  
L Trieschmann ◽  
Y V Postnikov ◽  
A Rickers ◽  
M Bustin
Keyword(s):  
Amino Acids ◽  
Structural Features ◽  
Binding Domain ◽  
Full Length ◽  
Modular Structure ◽  
Chromosomal Proteins ◽  
Nucleosome Core ◽  
Terminal Amino

Chromosomal proteins HMG-14 and HMG-17 are the only known nuclear proteins which specifically bind to the nucleosome core particle and are implicated in the generation and/or maintenance of structural features specific to active chromatin. The two proteins facilitate polymerase II and III transcription from in vitro- and in vivo-assembled circular chromatin templates. Here we used deletion mutants and specific peptides to identify the transcriptional enhancement domain and delineate the nucleosomal binding domain of the HMG-14 and -17 proteins. Deletion of the 22 C-terminal amino acids of HMG-17 or 26 C-terminal amino acids of HMG-14 reduces significantly the ability of the proteins to enhance transcription from chromatin templates. In contrast, N-terminal truncation mutants had the same transcriptional enhancement activity as the full-length proteins. We conclude that the negatively charged C-terminal region of the proteins is required for transcriptional enhancement. Chromatin transcription enhancement assays, which involve binding competition between the full-length proteins and peptides derived from their nucleosomal binding regions, indicate that the minimal nucleosomal binding domain of human HMG-17 is 24 amino acids long and spans residues 17 to 40. The results suggest that HMG-14 and -17 proteins have a modular structure and contain distinct functional domains.

scholarly journals Interaction of TATA-Binding Protein with Upstream Activation Factor Is Required for Activated Transcription of Ribosomal DNA by RNA Polymerase I in Saccharomyces cerevisiae In Vivo

1998 ◽  
Vol 18 (7) ◽  
pp. 3752-3761 ◽  
Author(s):  
Joan S. Steffan ◽  
Daniel A. Keys ◽  
Loan Vu ◽  
Masayasu Nomura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hybrid System ◽  
Ribosomal Dna ◽  
Tata Binding Protein ◽  
Rna Polymerase I ◽  
Activation Factor ◽  
Polymerase I ◽  
Two Hybrid

ABSTRACT Previous in vitro studies have shown that initiation of transcription of ribosomal DNA (rDNA) in the yeast Saccharomyces cerevisiae involves an interaction of upstream activation factor (UAF) with the upstream element of the promoter, forming a stable UAF-template complex; together with TATA-binding protein (TBP), UAF then recruits an essential factor, core factor (CF), to the promoter, forming a stable preinitiation complex. TBP interacts with both UAF and CF in vitro. In addition, a subunit of UAF, Rrn9p, interacts with TBP in vitro and in the two-hybrid system, suggesting the possible importance of this interaction for UAF function. Using the yeast two-hybrid system, we have identified three mutations inRRN9 that abolish the interaction of Rrn9p with TBP without affecting its interaction with Rrn10p, another subunit of UAF. Yeast cells containing any one of these individual mutations,L110S, L269P, or L274Q, did not show any growth defects. However, cells containing a combination ofL110S with one of the other two mutations showed a temperature-sensitive phenotype, and this phenotype was suppressed by fusing the mutant genes to SPT15, which encodes TBP. In addition, another mutation (F186S), which disrupts both Rrn9p-TBP and Rrn9p-Rrn10p interactions in the two-hybrid system, abolished UAF function in vivo, and this mutational defect was suppressed by fusion of the mutant gene to SPT15 combined with overexpression of Rrn10p. These experiments demonstrate that the interaction of UAF with TBP, which is presumably achieved by the interaction of Rrn9p with TBP, is indeed important for high-level transcription of rDNA by RNA polymerase I in vivo.

Calreticulin and Calreticulin Fragments Are Endothelial Cell Inhibitors That Suppress Tumor Growth

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2461-2468 ◽  
Author(s):  
Sandra E. Pike ◽  
Lei Yao ◽  
Joyce Setsuda ◽  
Karen D. Jones ◽  
Barry Cherney ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Proliferation ◽  
Endothelial Cell ◽  
Tumor Growth ◽  
Angiogenesis Inhibitors ◽  
Full Length ◽  
Endothelial Cell Proliferation ◽  
Proliferation In Vitro

Several angiogenesis inhibitors are fragments of larger proteins that are themselves not active as angiogenesis inhibitors. Vasostatin, the N-terminal domain of calreticulin inclusive of amino acids 1-180, is an angiogenesis inhibitor that exerts antitumor effects in vivo. In the present study, we examined whether the full-length calreticulin molecule shares the antiangiogenic and antitumor activities of vasostatin. Similar to vasostatin, calreticulin selectively inhibited endothelial cell proliferation in vitro, but not cells of other lineages, and suppressed angiogenesis in vivo. When inoculated into athymic mice, calreticulin inhibited Burkitt tumor growth comparably with vasostatin. Calreticulin lacking the N-terminal 1-120 amino acids inhibited endothelial cell proliferation in vitro and Burkitt tumor growth in vivo comparably with vasostatin. An internal calreticulin fragment encompassing amino acids 120-180 also inhibited endothelial cell proliferation in vitro and angiogenesis in vivo comparably with calreticulin and vasostatin. These results suggest that the antiangiogenic activities of vasostatin reside in a domain that is accessible from the full-length calreticulin molecule and localize to calreticulin N-terminal amino acids 120-180. Thus, calreticulin and calreticulin fragments are inhibitors of angiogenesis that directly target endothelial cells, inhibit angiogenesis, and suppress tumor growth. This information may be critical in designing targeted inhibitors of pathological angiogenesis that underlies cancer and other diseases.

scholarly journals The Biochemical Activities of the Saccharomyces cerevisiae Pif1 Helicase Are Regulated by Its N-Terminal Domain

Genes ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 411 ◽  
Author(s):  
Nickens ◽  
Sausen ◽  
Bochman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomerase Activity ◽  
Full Length ◽  
Dna Unwinding ◽  
Genome Maintenance ◽  
Terminal Domain ◽  
Pif1 Helicase ◽  
Telomerase Regulation

: Pif1 family helicases represent a highly conserved class of enzymes involved in multiple aspects of genome maintenance. Many Pif1 helicases are multi-domain proteins, but the functions of their non-helicase domains are poorly understood. Here, we characterized how the N-terminal domain (NTD) of the Saccharomyces cerevisiae Pif1 helicase affects its functions both in vivo and in vitro. Removal of the Pif1 NTD alleviated the toxicity associated with Pif1 overexpression in yeast. Biochemically, the N-terminally truncated Pif1 (Pif1ΔN) retained in vitro DNA binding, DNA unwinding, and telomerase regulation activities, but these activities differed markedly from those displayed by full-length recombinant Pif1. However, Pif1ΔN was still able to synergize with the Hrq1 helicase to inhibit telomerase activity in vitro, similar to full-length Pif1. These data impact our understanding of Pif1 helicase evolution and the roles of these enzymes in the maintenance of genome integrity.

scholarly journals Saccharomyces cerevisiae RAI1 (YGL246c) Is Homologous to Human DOM3Z and Encodes a Protein That Binds the Nuclear Exoribonuclease Rat1p

2000 ◽  
Vol 20 (11) ◽  
pp. 4006-4015 ◽  
Author(s):  
Yang Xue ◽  
Xinxue Bai ◽  
Insuk Lee ◽  
George Kallstrom ◽  
Jennifer Ho ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Rna Degradation ◽  
Growth Defect ◽  
Nuclear Targeting ◽  
Interacting Protein ◽  
Rrna Species ◽  
Synthetically Lethal

ABSTRACT The RAT1 gene of Saccharomyces cerevisiaeencodes a 5′→3′ exoribonuclease which plays an essential role in yeast RNA degradation and/or processing in the nucleus. We have cloned a previously uncharacterized gene (YGL246c) that we refer to asRAI1 (Rat1p interacting protein 1). RAI1 is homologous to Caenorhabditis elegans DOM-3 and humanDOM3Z. Deletion of RAI1 confers a growth defect which can be complemented by an additional copy of RAT1 on a centromeric vector or by directing Xrn1p, the cytoplasmic homolog of Rat1p, to the nucleus through the addition of a nuclear targeting sequence. Deletion of RAI1 is synthetically lethal with therat1-1ts mutation and shows genetic interaction with a deletion of SKI2 but not XRN1. Polysome analysis of an rai1 deletion mutant indicated a defect in 60S biogenesis which was nearly fully reversed by high-copyRAT1. Northern blot analysis of rRNAs revealed thatrai1 is required for normal 5.8S processing. In the absence of RAI1, 5.8SL was the predominant form of 5.8S and there was an accumulation of 3′-extended forms but not 5′-extended species of 5.8S. In addition, a 27S pre-rRNA species accumulated in therai1 mutant. Thus, deletion of RAI1 affects both 5′ and 3′ processing reactions of 5.8S rRNA. Consistent with the in vivo data suggesting that RAI1 enhances RAT1function, purified Rai1p stabilized the in vitro exoribonuclease activity of Rat1p.

scholarly journals Std1p (Msn3p) Positively Regulates the Snf1 Kinase in Saccharomyces cerevisiae

Genetics ◽  
2003 ◽  
Vol 163 (2) ◽  
pp. 507-514 ◽  
Author(s):  
Sergei Kuchin ◽  
Valmik K Vyas ◽  
Ellen Kanter ◽  
Seung-Pyo Hong ◽  
Marian Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Snf1 Kinase ◽  
Glucose Signaling ◽  
Catalytic Domains ◽  
Upstream Kinase ◽  
Two Hybrid

Abstract The Snf1 protein kinase of the glucose signaling pathway in Saccharomyces cerevisiae is regulated by an autoinhibitory interaction between the regulatory and catalytic domains of Snf1p. Transitions between the autoinhibited and active states are controlled by an upstream kinase and the Reg1p-Glc7p protein phosphatase 1. Previous studies suggested that Snf1 kinase activity is also modulated by Std1p (Msn3p), which interacts physically with Snf1p and also interacts with glucose sensors. Here we address the relationship between Std1p and the Snf1 kinase. Two-hybrid assays showed that Std1p interacts with the catalytic domain of Snf1p, and analysis of mutant kinases suggested that this interaction is incompatible with the autoinhibitory interaction of the regulatory and catalytic domains. Overexpression of Std1p increased the two-hybrid interaction of Snf1p with its activating subunit Snf4p, which is diagnostic of an open, uninhibited conformation of the kinase complex. Overexpression of Std1p elevated Snf1 kinase activity in both in vitro and in vivo assays. These findings suggest that Std1p stimulates the Snf1 kinase by an interaction with the catalytic domain that antagonizes autoinhibition and promotes an active conformation of the kinase.

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