Regulatory function of the Saccharomyces cerevisiae RAS C-terminus

Activating mutations (valine 19 or leucine 68) were introduced into the Saccharomyces cerevisiae RAS1 and RAS2 genes. In addition, a deletion was introduced into the wild-type gene and into an activated RAS2 gene, removing the segment of the coding region for the unique C-terminal domain that lies between the N-terminal 174 residues and the penultimate 8-residue membrane attachment site. At low levels of expression, a dominant activated phenotype, characterized by low glycogen levels and poor sporulation efficiency, was observed for both full-length RAS1 and RAS2 variants having impaired GTP hydrolytic activity. Lethal CDC25 mutations were bypassed by the expression of mutant RAS1 or RAS2 proteins with activating amino acid substitutions, by expression of RAS2 proteins lacking the C-terminal domain, or by normal and oncogenic mammalian Harvey ras proteins. Biochemical measurements of adenylate cyclase in membrane preparations showed that the expression of RAS2 proteins lacking the C-terminal domain can restore adenylate cyclase activity to cdc25 membranes.

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Characterization of TUP1, a mediator of glucose repression in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6500 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6500-6511 ◽

Cited By ~ 106

Author(s):

F E Williams ◽

R J Trumbly

Keyword(s):

Saccharomyces Cerevisiae ◽

Insertional Mutagenesis ◽

Glucose Repression ◽

Coding Region ◽

Gene Encoding ◽

Reading Frame ◽

C Terminus ◽

Single Deletion ◽

Rna Mapping ◽

Mutant Phenotypes

The TUP1 and CYC8 (= SSN6) genes of Saccharomyces cerevisiae play a major role in glucose repression. Mutations in either TUP1 or CYC8 eliminate or reduce glucose repression of many repressible genes and induce other phenotypes, including flocculence, failure to sporulate, and sterility of MAT alpha cells. The TUP1 gene was isolated in a screen for genes that regulate mating type (V.L. MacKay, Methods Enzymol. 101:325-343, 1983). We found that a 3.5-kb restriction fragment was sufficient for complete complementation of tup1-100. The gene was further localized by insertional mutagenesis and RNA mapping. Sequence analysis of 2.9 kb of DNA including TUP1 revealed only one long open reading frame which predicts a protein of molecular weight 78,221. The predicted protein is rich in serine, threonine, and glutamine. In the carboxyl region there are six repeats of a pattern of about 43 amino acids. This same pattern of conserved residues is seen in the beta subunit of transducin and the yeast CDC4 gene product. Insertion and deletion mutants are viable, with the same range of phenotypes as for point mutants. Deletions of the 3' end of the coding region produced the same mutant phenotypes as did total deletions, suggesting that the C terminus is critical for TUP1 function. Strains with deletions in both the CYC8 and TUP1 genes are viable, with phenotypes similar to those of strains with a single deletion. A deletion mutation of TUP1 was able to suppress the snf1 mutation block on expression of the SUC2 gene encoding invertase.

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Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5' coding region of the adenylate cyclase gene

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5555-5560.1988 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5555-5560

Author(s):

H Iida

Keyword(s):

Heat Treatment ◽

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Adenylate Cyclase ◽

Uv Light ◽

Yeast Cells ◽

Wild Type ◽

Coding Region ◽

Resistant Mutants ◽

Type Strains

Heat shock-resistant mutants, which were isolated by their ability to withstand lethal heat treatment, were characterized. Resistance was demonstrated to be a consequence of insertion of retrotransposon Ty into either the 5' coding or noncoding region, close to the putative initiation codon of the adenylate cyclase gene CYR1 (or CDC35). These heat shock-resistant mutants contained about threefold lower adenylate cyclase activity than wild-type strains. The mutants were also observed to be resistant to other stresses such as UV light and ethanol. These results demonstrate that multistress resistance, which may confer a survival advantage to yeast cells, can be generated by transposition of a Ty element into CYR1.

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Fip1 Regulates the Activity of Poly(A) Polymerase through Multiple Interactions

Molecular and Cellular Biology ◽

10.1128/mcb.21.6.2026-2037.2001 ◽

2001 ◽

Vol 21 (6) ◽

pp. 2026-2037 ◽

Cited By ~ 35

Author(s):

Steffen Helmling ◽

Alexander Zhelkovsky ◽

Claire L. Moore

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Rna Binding ◽

Physiological Function ◽

Regulatory Function ◽

Functional Domains ◽

Essential Component ◽

C Terminus ◽

Mrna Precursor ◽

Multiple Interactions

ABSTRACT Fip1 is an essential component of the Saccharomyces cerevisiae polyadenylation machinery and the only protein known to interact directly with poly(A) polymerase (Pap1). Its association with Pap1 inhibits the extension of an oligo(A) primer by limiting access of the RNA substrate to the C-terminal RNA binding domain (C-RBD) of Pap1. We present here the identification of separate functional domains of Fip1. Amino acids 80 to 105 are required for binding to Pap1 and for the inhibition of Pap1 activity. This region is also essential for viability, suggesting that Fip1-mediated repression of Pap1 has a crucial physiological function. Amino acids 206 to 220 of Fip1 are needed for the interaction with the Yth1 subunit of the complex and for specific polyadenylation of the cleaved mRNA precursor. A third domain within amino acids 105 to 206 helps to limit RNA binding at the C-RBD of Pap1. Our data demonstrate that the C terminus of Fip1 is required to relieve the Fip1-mediated repression of Pap1 in specific polyadenylation. In the absence of this domain, Pap1 remains in an inhibited state. These findings show that Fip1 has a crucial regulatory function in the polyadenylation reaction by controlling the activity of poly(A) tail synthesis through multiple interactions within the polyadenylation complex.

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Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5' coding region of the adenylate cyclase gene.

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5555 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5555-5560 ◽

Cited By ~ 41

Author(s):

H Iida

Keyword(s):

Heat Treatment ◽

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Adenylate Cyclase ◽

Uv Light ◽

Yeast Cells ◽

Wild Type ◽

Coding Region ◽

Resistant Mutants ◽

Type Strains

Heat shock-resistant mutants, which were isolated by their ability to withstand lethal heat treatment, were characterized. Resistance was demonstrated to be a consequence of insertion of retrotransposon Ty into either the 5' coding or noncoding region, close to the putative initiation codon of the adenylate cyclase gene CYR1 (or CDC35). These heat shock-resistant mutants contained about threefold lower adenylate cyclase activity than wild-type strains. The mutants were also observed to be resistant to other stresses such as UV light and ethanol. These results demonstrate that multistress resistance, which may confer a survival advantage to yeast cells, can be generated by transposition of a Ty element into CYR1.

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Conserved luminal C-terminal domain dynamically controls interdomain communication in sarcolipin

10.1101/2020.03.28.013425 ◽

2020 ◽

Cited By ~ 1

Author(s):

Rodrigo Aguayo-Ortiz ◽

Eli Fernández-de Gortari ◽

L. Michel Espinoza-Fonseca

Keyword(s):

Functional Divergence ◽

Transmembrane Helix ◽

Dynamic Control ◽

Essential Element ◽

Md Simulations ◽

Regulatory Function ◽

Functional Feature ◽

Terminal Domain ◽

C Terminus ◽

Interdomain Communication

ABSTRACTSarcolipin (SLN) mediates Ca2+ transport and metabolism in muscle by regulating the activity of the Ca2+ pump SERCA. SLN has a conserved luminal C-terminal domain that contributes to the its functional divergence among homologous SERCA regulators, but the precise mechanistic role of this domain remains poorly understood. We used all-atom molecular dynamics (MD) simulations of SLN totaling 77.5 μs to show that the N- (NT) and C-terminal (CT) domains function in concert. Analysis of the MD simulations showed that serial deletions of SLN C-terminus does not affect the stability of the peptide nor induce dissociation of SLN from the membrane but promotes a gradual decrease in both tilt angle of the transmembrane helix and the local thickness of the lipid bilayer. Mutual information analysis showed that the NT and CT domains communicate with each other in SLN, and that interdomain communication is partially or completely abolished upon deletion of the conserved segment Tyr29-Tyr31 as well as by serial deletions beyond this domain. Phosphorylation of SLN at residue Thr5 also induces changes in the communication between the CT and NT domains, thus providing additional evidence for interdomain communication within SLN. We found that interdomain communication is independent of the force field used and lipid composition, thus demonstrating that communication between the NT and CT domains is an intrinsic functional feature of SLN. We propose the novel hypothesis that the conserved C-terminus is an essential element required for dynamic control of SLN regulatory function.Abstract Figure

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Interactions of Sen1, Nrd1, and Nab3 with Multiple Phosphorylated Forms of the Rpb1 C-Terminal Domain in Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.05320-11 ◽

2012 ◽

Vol 11 (4) ◽

pp. 417-429 ◽

Cited By ~ 39

Author(s):

Karen Chinchilla ◽

Juan B. Rodriguez-Molina ◽

Doris Ursic ◽

Jonathan S. Finkel ◽

Aseem Z. Ansari ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase Ii ◽

Protein Interactions ◽

Noncoding Rna ◽

Protein Protein Interactions ◽

Coding Region ◽

Protein Coding ◽

Content Type ◽

Protein Coding Genes ◽

Terminal Domain

ABSTRACT The Saccharomyces cerevisiae SEN1 gene codes for a nuclear, ATP-dependent helicase which is embedded in a complex network of protein-protein interactions. Pleiotropic phenotypes of mutations in SEN1 suggest that Sen1 functions in many nuclear processes, including transcription termination, DNA repair, and RNA processing. Sen1, along with termination factors Nrd1 and Nab3, is required for the termination of noncoding RNA transcripts, but Sen1 is associated during transcription with coding and noncoding genes. Sen1 and Nrd1 both interact directly with Nab3, as well as with the C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II. It has been proposed that Sen1, Nab3, and Nrd1 form a complex that associates with Rpb1 through an interaction between Nrd1 and the Ser 5 -phosphorylated (Ser 5 -P) CTD. To further study the relationship between the termination factors and Rpb1, we used two-hybrid analysis and immunoprecipitation to characterize sen1-R302W , a mutation that impairs an interaction between Sen1 and the Ser 2 -phosphorylated CTD. Chromatin immunoprecipitation indicates that the impairment of the interaction between Sen1 and Ser 2 -P causes the reduced occupancy of mutant Sen1 across the entire length of noncoding genes. For protein-coding genes, mutant Sen1 occupancy is reduced early and late in transcription but is similar to that of the wild type across most of the coding region. The combined data suggest a handoff model in which proteins differentially transfer from the Ser 5 - to the Ser 2 -phosphorylated CTD to promote the termination of noncoding transcripts or other cotranscriptional events for protein-coding genes.

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The C-Terminal Domain of Sin1 Interacts with the SWI-SNF Complex in Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.18.7.4157 ◽

1998 ◽

Vol 18 (7) ◽

pp. 4157-4164 ◽

Cited By ~ 21

Author(s):

José Pérez-Martín ◽

Alexander D. Johnson

Keyword(s):

Saccharomyces Cerevisiae ◽

Histone H3 ◽

Full Length ◽

Terminal Domain ◽

C Terminus

ABSTRACT In the yeast Saccharomyces cerevisiae, the SWI-SNF complex has been proposed to antagonize the repressive effects of chromatin by disrupting nucleosomes. The SIN genes were identified as suppressors of defects in the SWI-SNF complex, and theSIN1 gene encodes an HMG1-like protein that has been proposed to be a component of chromatin. Specific mutations (sin mutations) in both histone H3 and H4 genes produce the same phenotypic effects as do mutations in the SIN1 gene. In this study, we demonstrate that Sin1 and the H3 and H4 histones interact genetically and that the C terminus of Sin1 physically associates with components of the SWI-SNF complex. In addition, we demonstrate that this interaction is blocked in the full-length Sin1 protein by the N-terminal half of the protein. Based on these and additional results, we propose that Sin1 acts as a regulatable bridge between the SWI-SNF complex and the nucleosome.

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Phytochrome and the regulation of the expression of its genes

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1986.0066 ◽

1986 ◽

Vol 314 (1166) ◽

pp. 469-480 ◽

Cited By ~ 37

Keyword(s):

Conformational Changes ◽

Attachment Site ◽

Terminal Segment ◽

Amino Acid Sequences ◽

Regulatory Function ◽

Nucleotide Sequence Analysis ◽

Mrna Levels ◽

Hydrophobic Region ◽

Terminal Domain ◽

Run Off

In attempting to understand the mechanism of phytochrome action we are studying structural properties of the photoreceptor molecule and the autoregulation of expression of phytochrome genes. Run-off transcription assays in isolated nuclei from Avena indicate that phytochrome decreases the transcription of its own genes threefold in less than 15 min from Pfr formation. The extent of this decrease is insufficient to account for the observed 10- to 50-fold decrease in mature phytochrome mRNA levels, suggesting that enhanced degradation may also play a significant role in determining the level of this mRNA. Structural analysis of native phytochrome from Avena indicates that the molecule is an elongated dimer of 124 kDa monomers, each consisting of a globular, 74 kDa, NH 2 -terminal domain bearing the single chromophore at Cys-321, and a more open COOH-terminal domain that bears the dimerization site. Controlled proteolysis and binding of monoclonal antibodies to mapped epitopes has identified two regions, one in the 6-10 kDa NH 2 -terminal segment and the other ca. 70 kDa from the NH 2 -terminus, that undergo photoconversion-induced conformational changes and are therefore candidates for involvement in the molecule’s regulatory function. Comparison of the full-length amino acid sequences of Avena and Cucurbita phytochromes, derived from nucleotide sequence analysis, indicates overall homology of 65%. The most highly conserved regions are those immediately surrounding the chromophore attachment site, where 29 residues are invariant, and a hydrophobic region between residues 150 and 300, postulated to form a cavity containing the chromophore. In contrast, a strikingly lower level of homology exists at the COOH-terminus of the polypeptide between residues 800 and 1128, indicating a possible lack of involvement of this region in phytochrome function.

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