scholarly journals Myb-Related Schizosaccharomyces pombecdc5p Is Structurally and Functionally Conserved in Eukaryotes

1998 ◽  
Vol 18 (7) ◽  
pp. 4097-4108 ◽  
Author(s):  
Ryoma Ohi ◽  
Anna Feoktistova ◽  
Stacey McCann ◽  
Virginia Valentine ◽  
A. Thomas Look ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drosophila Melanogaster ◽  
Transcriptional Activation ◽  
Homo Sapiens ◽  
Family Members ◽  
Temperature Sensitive ◽  
Related Protein ◽  
Hydrophobic Residues ◽  
Functional Conservation

ABSTRACT Schizosaccharomyces pombe cdc5p is a Myb-related protein that is essential for G2/M progression. To explore the structural and functional conservation of Cdc5 throughout evolution, we isolated Cdc5-related genes and cDNAs fromSaccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens. Supporting the notion that these Cdc5 gene family members are functionally homologous to S. pombe cdc5 +, human and fly Cdc5 cDNAs are capable of complementing the temperature-sensitive lethality of the S. pombe cdc5-120 mutant. Furthermore, S. cerevisiae CEF1(S. cerevisiae homolog of cdc5 +), like S. pombe cdc5 +, is essential during G2/M. The location of the cdc5-120 mutation, as well as mutational analyses of Cef1p, indicate that the Myb repeats of cdc5p and Cef1p are important for their function in vivo. However, we found that unlike in c-Myb, single residue substitutions of glycines for hydrophobic residues within the Myb repeats of Cef1p, which are essential for maintaining structure of the Myb domain, did not impair Cef1p function in vivo. Rather, multiple W-to-G substitutions were required to inactivate Cef1p, and many of the substitution mutants were found to confer temperature sensitivity. Although it is possible that Cef1p acts as a transcriptional activator, we have demonstrated that Cef1p is not involved in transcriptional activation of a class of G2/M-regulated genes typified by SWI5. Collectively, these results suggest that Cdc5 family members participate in a novel pathway to regulate G2/M progression.

Functional conservation of tRNase ZL among Saccharomyces cerevisiae, Schizosaccharomyces pombe and humans

2009 ◽  
Vol 422 (3) ◽  
pp. 483-492 ◽  
Author(s):  
Zhen Zhao ◽  
Wenchen Su ◽  
Sheng Yuan ◽  
Ying Huang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Schizosaccharomyces Pombe ◽  
Cancer Susceptibility ◽  
Short Form ◽  
Susceptibility Gene ◽  
Temperature Sensitive ◽  
Functional Conservation ◽  
Trnase Z

Although tRNase Z from various organisms was shown to process nuclear tRNA 3′ ends in vitro, only a very limited number of studies have reported its in vivo biological functions. tRNase Z is present in a short form, tRNase ZS, and a long form, tRNase ZL. Unlike Saccharomyces cerevisiae, which contains one tRNase ZL gene (scTRZ1) and humans, which contain one tRNase ZL encoded by the prostate-cancer susceptibility gene ELAC2 and one tRNase ZS, Schizosaccharomyces pombe contains two tRNase ZL genes, designated sptrz1+ and sptrz2+. We report that both sptrz1+ and sptrz2+ are essential for growth. Moreover, sptrz1+ is required for cell viability in the absence of Sla1p, which is thought to be required for endonuclease-mediated maturation of pre-tRNA 3′ ends in yeast. Both scTRZ1 and ELAC2 can complement a temperature-sensitive allele of sptrz1+, sptrz1–1, but not the sptrz1 null mutant, indicating that despite exhibiting species specificity, tRNase ZLs are functionally conserved among S. cerevisiae, S. pombe and humans. Overexpression of sptrz1+, scTRZ1 and ELAC2 can increase suppression of the UGA nonsense mutation ade6–704 through facilitating 3′ end processing of the defective suppressor tRNA that mediates suppression. Our findings reveal that 3′ end processing is a limiting step for defective tRNA maturation and demonstrate that overexpression of sptrz1+, scTRZ1 and ELAC2 can promote defective tRNA 3′ processing in vivo. Our results also support the notion that yeast tRNase ZL is absolutely required for 3′ end processing of at least a few pre-tRNAs even in the absence of Sla1p.

scholarly journals Functional Conservation of the Glutamine-Rich Domains of Yeast Gal11 and Human SRC-1 in the Transactivation of Glucocorticoid Receptor Tau 1 in Saccharomyces cerevisiae

2007 ◽  
Vol 28 (3) ◽  
pp. 913-925 ◽  
Author(s):  
Dae-Hwan Kim ◽  
Gwang Sik Kim ◽  
Chul Ho Yun ◽  
Young Chul Lee
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Mediator Complex ◽  
Dependent Manner ◽  
Activator Proteins ◽  
Functional Conservation

ABSTRACT The yeast Gal11 protein, a component of the Mediator complex, is required for the transcriptional activation of many class II genes as a physiological target of various activator proteins in vivo. In this study, we identified the yeast (Saccharomyces cerevisiae) Mediator complex as a novel coactivator of the transcriptional activity of the glucocorticoid receptor (GR) tau 1 (τ1), the major transcriptional activation domain of the GR. GR τ1 directly interacted with the Mediator complex in vivo and in vitro in a Gal11 module-dependent manner, and the Gal11p subunit interacted directly with GR τ1. Specific amino acid residues within the glutamine-rich (Qr) domain of Gal11p (residues 116 to 277) were essential for its interaction with GR τ1 and GR τ1 transactivity in yeast, as demonstrated by mutational analysis of the Gal11 Qr domain, which is highly conserved among human steroid receptor coactivator (SRC) proteins. A Gal11p variant, mini-Gal11p, comprised of the Mediator association and Qr domains of Gal11p or chimeric mini-Gal11p containing the Qr domain of SRC-1 could potentiate the GR τ1 transactivity in a gal11Δ yeast strain. These results suggest that there is functional conservation between Qr domains of yeast Gal11p and mammalian SRC proteins as direct targets of activator proteins in yeast.

scholarly journals Mapping of a Yeast G Protein βγ Signaling Interaction

Genetics ◽  
1998 ◽  
Vol 150 (4) ◽  
pp. 1407-1417 ◽  
Author(s):  
Simon J Dowell ◽  
Anne L Bishop ◽  
Susan L Dyos ◽  
Andrew J Brown ◽  
Malcolm S Whiteway
Keyword(s):  
Signal Transduction ◽  
Map Kinase ◽  
G Protein ◽  
Coiled Coil ◽  
Receptor Activation ◽  
Temperature Sensitive ◽  
Hydrophobic Residues ◽  
Map Kinase Cascade ◽  
Kinase Cascade

Abstract The mating pathway of Saccharomyces cerevisiae is widely used as a model system for G protein-coupled receptor-mediated signal transduction. Following receptor activation by the binding of mating pheromones, G protein βγ subunits transmit the signal to a MAP kinase cascade, which involves interaction of Gβ (Ste4p) with the MAP kinase scaffold protein Ste5p. Here, we identify residues in Ste4p required for the interaction with Ste5p. These residues define a new signaling interface close to the Ste20p binding site within the Gβγ coiled-coil. Ste4p mutants defective in the Ste5p interaction interact efficiently with Gpa1p (Gα) and Ste18p (Gγ) but cannot function in signal transduction because cells expressing these mutants are sterile. Ste4 L65S is temperature-sensitive for its interaction with Ste5p, and also for signaling. We have identified a Ste5p mutant (L196A) that displays a synthetic interaction defect with Ste4 L65S, providing strong evidence that Ste4p and Ste5p interact directly in vivo through an interface that involves hydrophobic residues. The correlation between disruption of the Ste4p-Ste5p interaction and sterility confirms the importance of this interaction in signal transduction. Identification of the Gβγ coiled-coil in Ste5p binding may set a precedent for Gβγ-effector interactions in more complex organisms.

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

Interspersion of an unusual GCN4 activation site with a complex transcriptional repression site in Ty2 elements of Saccharomyces cerevisiae

1993 ◽  
Vol 13 (4) ◽  
pp. 2091-2103
Author(s):  
S Türkel ◽  
P J Farabaugh
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Physiological Control ◽  
Reading Frame ◽  
Negative Region ◽  
Activation Site

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes at least two downstream activation sites (DASs). This paper concerns the characterization of DASI. A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation. We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell. We have termed the two sites GCN4 binding sites (GBS1 and GBS2). GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site. GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity. Nucleotides 13 to 36 of DASI overlap DRSII. Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements. We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4). One of these is repeated in the 12 bp immediately adjacent to DASI. Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators. The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.

scholarly journals Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases.

1991 ◽  
Vol 11 (7) ◽  
pp. 3463-3471 ◽  
Author(s):  
S R Schmid ◽  
P Linder
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Clear Correlation ◽  
Temperature Sensitive ◽  
Rna Helicases ◽  
Initiation Of Translation

The eukaryotic translation initiation factor 4A (eIF-4A) possesses an in vitro helicase activity that allows the unwinding of double-stranded RNA. This activity is dependent on ATP hydrolysis and the presence of another translation initiation factor, eIF-4B. These two initiation factors are thought to unwind mRNA secondary structures in preparation for ribosome binding and initiation of translation. To further characterize the function of eIF-4A in cellular translation and its interaction with other elements of the translation machinery, we have isolated mutations in the TIF1 and TIF2 genes encoding eIF-4A in Saccharomyces cerevisiae. We show that three highly conserved domains of the D-E-A-D protein family, encoding eIF-4A and other RNA helicases, are essential for protein function. Only in rare cases could we make a conservative substitution without affecting cell growth. The mutants show a clear correlation between their growth and in vivo translation rates. One mutation that results in a temperature-sensitive phenotype reveals an immediate decrease in translation activity following a shift to the nonpermissive temperature. These in vivo results confirm previous in vitro data demonstrating an absolute dependence of translation on the TIF1 and TIF2 gene products.

scholarly journals Extensive mutagenesis of a transcriptional activation domain identifies single hydrophobic and acidic amino acids important for activation in vivo.

1997 ◽  
Vol 17 (1) ◽  
pp. 115-122 ◽  
Author(s):  
M B Sainz ◽  
S A Goff ◽  
V L Chandler
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transcriptional Activation ◽  
Carboxyl Terminus ◽  
Wild Type ◽  
Activation Domain ◽  
Hydrophobic Residues ◽  
Transcriptional Activation Domain ◽  
Acidic Amino Acids

C1 is a transcriptional activator of genes encoding biosynthetic enzymes of the maize anthocyanin pigment pathway. C1 has an amino terminus homologous to Myb DNA-binding domains and an acidic carboxyl terminus that is a transcriptional activation domain in maize and yeast cells. To identify amino acids critical for transcriptional activation, an extensive random mutagenesis of the C1 carboxyl terminus was done. The C1 activation domain is remarkably tolerant of amino acid substitutions, as changes at 34 residues had little or no effect on transcriptional activity. These changes include introduction of helix-incompatible amino acids throughout the C1 activation domain and alteration of most single acidic amino acids, suggesting that a previously postulated amphipathic alpha-helix is not required for activation. Substitutions at two positions revealed amino acids important for transcriptional activation. Replacement of leucine 253 with a proline or glutamine resulted in approximately 10% of wild-type transcriptional activation. Leucine 253 is in a region of C1 in which several hydrophobic residues align with residues important for transcriptional activation by the herpes simplex virus VP16 protein. However, changes at all other hydrophobic residues in C1 indicate that none are critical for C1 transcriptional activation. The other important amino acid in C1 is aspartate 262, as a change to valine resulted in only 24% of wild-type transcriptional activation. Comparison of our C1 results with those from VP16 reveal substantial differences in which amino acids are required for transcriptional activation in vivo by these two acidic activation domains.

An essential Saccharomyces cerevisiae gene homologous to SNF2 encodes a helicase-related protein in a new family

1992 ◽  
Vol 12 (4) ◽  
pp. 1893-1902
Author(s):  
B C Laurent ◽  
X Yang ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Nucleoside Triphosphate ◽  
Related Protein ◽  
New Family ◽  
Eukaryotic Proteins ◽  
Beta Galactosidase

The Saccharomyces cerevisiae SNF2 gene affects the expression of many diversely regulated genes and has been implicated in transcriptional activation. We report here the cloning and characterization of STH1, a gene that is homologous to SNF2. STH1 is essential for mitotic growth and is functionally distinct from SNF2. A bifunctional STH1-beta-galactosidase protein is located in the nucleus. The predicted 155,914-Da STH1 protein is 72% identical to SNF2 over 661 amino acids and 46% identical over another stretch of 66 amino acids. Both STH1 and SNF2 contain a putative nucleoside triphosphate-binding site and sequences resembling the consensus helicase motifs. The large region of homology shared by STH1 and SNF2 is conserved among other eukaryotic proteins, and STH1 and SNF2 appear to define a novel family of proteins related to helicases.

scholarly journals Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis.

1997 ◽  
Vol 17 (9) ◽  
pp. 5001-5015 ◽  
Author(s):  
N I Zanchin ◽  
P Roberts ◽  
A DeSilva ◽  
F Sherman ◽  
D S Goldfarb
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Open Reading Frames ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Protein Fusion ◽  
Acid Protein ◽  
Conserved Gene ◽  
Green Fluorescent

The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.

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