Role of Sch9 in regulating Ras-cAMP signal pathway in Saccharomyces cerevisiae

In Saccharomyces cerevisiae PKA plays a major role in regulating cell growth, metabolism, and stress resistance. We report that Sch9 regulates PKA directly and SCH9 deletion enhances PKA activity by showing that: (1) Bcy1 predominately localized in the nucleus in glycerol-grown sch9Δ cells; (2) large part of the catalytic subunits of PKA transferred from the nucleus to the cytoplasm in sch9Δ cells; (3) higher protein stability of Tpk2 resulted in higher protein level of Tpk2 in sch9Δ than in wild type cells. Our investigations suggest that Sch9 regulates phosphorylation of Bcy1. We also observed hyper-phosphorylation of Cdc25 in sch9Δ, in contrast to the tpk2Δ and tpk2Δsch9Δ mutants, suggesting that feedback inhibition of PKA on Cdc25 is through Tpk2.     

RNA and protein elongation rates in Saccharomyces cerevisiae

Summary The RNA elongation rate has been measured in yeast by the kinetics of appearance of radioactivity in the different molecular weight classes by the method first developed by Bremer and Yuan (1968). Despite the limitations caused by the breakdown of the 35s rRNA precursor, an estimate of 29 to 38 nucleotides/second at 30° has been obtained for the RNA elongation rate. The protein elongation rate has been calculated by the method of Maaløe and Kjeldgaard (1966) which consists of dividing the number of amino acids polymerized into protein per unit of time by the number of active ribosomes. This has given values of 7 to 9 amino acids/second at 30°.These numbers are of the same order as those found in Escherichia coli when corrected to 37°. Eucaryotic cells could thus have preserved part of the coupling found in bacteria between RNA and protein elongation rates.     

Mitochondrial translational-initiation and elongation factors in Saccharomyces cerevisiae

C155 and E252 are respiratory-defective mutants of Saccharomyces cerevisiae, previously assigned to complementation groups G37 and G142, respectively. The following evidence suggested that both mutants were likely to have lesions in components of the mitochondrial translational machinery: C155 and E252 display a pleiotropic deficiency in cytochromes a, a3 and b; both strains are severly limited in their ability to incorporate radioactive methionine into the mitochondrial translation products and, in addition, display a tendency to loose wild-type mitochondrial DNA. This set of characteristics is commonly found in strains affected in mitochondrial protein synthesis. To identify the biochemical lesions, each mutant was transformed with a wild-type yeast genomic library and clones complemented for the respiratory defect were selected for growth on a non-fermentable substrate. Analysis of the cloned genes revealed that C155 has a mutation in a protein which has high sequence similarity to bacterial elongation factor G and that E252 has a mutation in a protein homologous to bacterial initiation factor 2. Disruption of the chromosomal copy of each gene in a wild-type haploid yeast induced a phenotype analogous to that of the original mutants, but does not affect cell viability. These results indicate that both gene products function exclusively in mitochondrial protein synthesis. Subcloning of the IFM1 gene, coding for the mitochondrial initiation factor, indicates that the amino-terminal 423 residues of the protein are sufficient to promote peptide-chain initiation in vivo.     

Pol3 is involved in nonhomologous end-joining in Saccharomyces cerevisiae

Nonhomologous end joining connects DNA ends in the absence of extended sequence homology and requires removal of mismatched DNA ends and gap-filling synthesis prior to a religation step. Pol4 within the Pol X family is the only polymerase known to be involved in end processing during nonhomologous end joining in yeast. The Saccharomyces cerevisiae POL3/CDC2 gene encodes polymerase delta that is involved in DNA replication and other DNA repair processes. Here, we show that POL3 is involved in nonhomologous end joining using a plasmid-based end-joining assay in yeast, in which the pol3-t mutation caused a 1.9- to 3.2-fold decrease in the end-joining efficiency of partially compatible 5' or 3' ends, or incompatible ends, similar to the pol4 mutant. The pol3-t pol4 double mutation showed a synergistic decrease in the efficiency of NHEJ with partially compatible 5' ends or incompatible ends. Sequence analysis of the rejoined junctions recovered from the wild-type cells and mutants indicated that POL3 is required for gap filling at 3' overhangs, but not 5' overhangs during POL4-independent nonhomologous end joining. We also show that either Pol3 or Pol4 is required for simple religation of compatible or blunt ends. These results suggest that Pol3 has a generalized function in end joining in addition to its role in gap filling at 3' overhangs to enhance the overall efficiency of nonhomologous end joining. Moreover, the decreased end-joining efficiency seen in the pol3-t mutant was not due to S-phase arrest associated with the mutant. Taken together, our genetic evidence supports a novel role of Pol3 in nonhomologous end joining that facilitates gap filling at 3' overhangs in the absence of Pol4 to maintain genomic integrity.     

RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach

To physically characterize the web of interactions connecting the Saccharomyces cerevisiae proteins suspected to be RNA polymerase II (RNAPII) elongation factors, subunits of Spt4/Spt5 and Spt16/Pob3 (corresponding to human DSIF and FACT), Spt6, TFIIF (Tfg1, -2, and -3), TFIIS, Rtf1, and Elongator (Elp1, -2, -3, -4, -5, and -6) were affinity purified under conditions designed to minimize loss of associated polypeptides and then identified by mass spectrometry. Spt16/Pob3 was discovered to associate with three distinct complexes: histones; Chd1/casein kinase II (CKII); and Rtf1, Paf1, Ctr9, Cdc73, and a previously uncharacterized protein, Leo1. Rtf1 and Chd1 have previously been implicated in the control of elongation, and the sensitivity to 6-azauracil of strains lacking Paf1, Cdc73, or Leo1 suggested that these proteins are involved in elongation by RNAPII as well. Confirmation came from chromatin immunoprecipitation (ChIP) assays demonstrating that all components of this complex, including Leo1, cross-linked to the promoter, coding region, and 3' end of the ADH1 gene. In contrast, the three subunits of TFIIF cross-linked only to the promoter-containing fragment of ADH1. Spt6 interacted with the uncharacterized, essential protein Iws1 (interacts with Spt6), and Spt5 interacted either with Spt4 or with a truncated form of Spt6. ChIP on Spt6 and the novel protein Iws1 resulted in the cross-linking of both proteins to all three regions of the ADH1 gene, suggesting that Iws1 is likely an Spt6-interacting elongation factor. Spt5, Spt6, and Iws1 are phosphorylated on consensus CKII sites in vivo, conceivably by the Chd1/CKII associated with Spt16/Pob3. All the elongation factors but Elongator copurified with RNAPII.     

Prediction of nucleosome occupancy in Saccharomyces cerevisiae using position-correlation scoring function

Knowledge of the detailed organization of nucleosomes across genomes and the mechanisms of nucleosome positioning is critical for the understanding of gene regulation and expression. In the present work, the bias of 4-mer frequency in nucleosome and linker sequences of the S. cerevisiae genome was analyzed statistically. A novel position-correlation scoring function algorithm based on the bias of 4-mer frequency in linker sequences was presented to distinguish nucleosome vs linker sequences. Five-fold cross-validation demonstrated that the algorithm achieved a good performance with mean area under the receiver operator characteristics curve of 0.981. Next, the algorithm was used to predict nucleosome occupancy throughout the S. cerevisiae genome and relatively high correlation coefficients with experiment maps of nucleosome positioning were obtained. Besides, the distinct nucleosome depleted regions in the vicinity of regulatory sites were confirmed. The results suggest that intrinsic DNA sequence preferences in linker regions have a significant impact on the nucleosome occupancy.     

Isolation and properties of elongation factor 1 from Saccharomyces cerevisiae

Polypeptide elongation factor 1 was isolated from yeast postribosomal supernatant. The highly purified factor was resolved on Ultrogel AcA-44 into two complementary fractions. One of these fractions contained two different polypeptide chains corresponding to a Ts-like elongation factor EF-1 beta gamma. The other fraction represented the light form of the factor, designated EF-1 alpha, with a molecular weight of approximately 50,000. The obtained results indicate that EF-1 from lower eukaryotes is also composed of three distinct polypeptides.     

Purification and characterization of Saccharomyces cerevisiae mitochondrial elongation factor Tu

Yeast mitochondrial elongation factor Tu (EF-Tu) was purified 200-fold from a mitochondrial extract of Saccharomyces cerevisiae to yield a single polypeptide of Mr = approximately 47,000. The factor was detected by complementation with Escherichia coli elongation factor G and ribosomes in an in vitro phenylalanine polymerization reaction. Mitochondrial EF-Tu, like E. coli EF-Tu, catalyzes the binding of aminoacyl-tRNA to ribosomes and possesses an intrinsic GTP hydrolyzing activity which can be activated either by kirromycin or by ribosomes. Kinetic and binding analyses of the interactions of mitochondrial EF-Tu with guanine nucleotides yielded affinity constants for GTP and GDP of approximately 5 and 25 microM, respectively. The corresponding affinity constants for the E. coli factor are approximately 0.3 and 0.003 microM, respectively. In keeping with these observations, we found that purified mitochondrial EF-Tu, unlike E. coli EF-Tu, does not contain endogenously bound nucleotide and is not stabilized by GDP. In addition, we have been unable to detect a functional counterpart to E. coli EF-Ts in extracts of yeast mitochondria and E. coli EF-Ts did not detectably stimulate amino acid polymerization with mitochondrial EF-Tu or enhance the binding of guanine nucleotides to the factor. We conclude that while yeast mitochondrial EF-Tu is functionally analogous to and interchangeable with E. coli EF-Tu, its affinity for guanine nucleotides and interaction with EF-Ts are quite different from those of E. coli EF-Tu.     

The importance of post-translational modifications in regulating Saccharomyces cerevisiae metabolism

Regulation of the flow of mass and energy through cellular metabolic networks is fundamental to the operation of all living organisms. Such metabolic fluxes are determined by the concentration of limiting substrates and by the amount and kinetic properties of the enzymes. Regulation of the amount of enzyme can be exerted, on a long-term scale, at the level of gene and protein expression. Enzyme regulation by post-translational modifications (PTMs) and noncovalent binding of allosteric effectors are shorter-term mechanisms that modulate enzyme activity. PTMs, in particular protein phosphorylation, are increasingly being recognized as key regulators in many cellular processes, including metabolism. For example, about half of the enzymes in the Saccharomyces cerevisiae metabolic network have been detected as phosphoproteins, although functional relevance has been demonstrated only in a few cases. Direct regulation of enzymes by PTMs provides one of the fastest ways for cells to adjust to environmental cues and internal stimulus. This review charts the so far identified metabolic enzymes undergoing reversible PTMs in the model eukaryote S. cerevisiae and reviews their underlying mechanistic principles - both at the individual enzyme level and in the context of the entire metabolic network operation.     

Signal transduction cascades regulating pseudohyphal differentiation of Saccharomyces cerevisiae

In response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous pseudohyphal growth. At least two signaling pathways regulate filamentation. One involves components of the MAP kinase cascade that also regulates mating of haploid cells. The second involves a nutrient-sensing G-protein-coupled receptor that signals via an unusual G(alpha) protein, cAMP and protein kinase A. Recent studies reveal crosstalk between these pathways during pseudohyphal growth. Related MAP kinase and cAMP pathways regulate filamentation and virulence of human and plant fungal pathogens, and represent novel targets for antifungal drug design.     

The effect of oxidative metabolism on spontaneous Pol zeta-dependent translesion synthesis in Saccharomyces cerevisiae

DNA lesions can stall or block high-fidelity polymerases, thus inhibiting replication. To bypass such lesions, low-fidelity translesion synthesis (TLS) polymerases can be used to insert a nucleotide across from the lesion or extend from a lesion:base mispair. When DNA repair is compromised in Saccharomyces cerevisiae, spontaneous DNA lesions can lead to a novel mutational event in which a frameshift is accompanied by one or more base pair substitutions. These "complex frameshifts" are dependent upon the TLS polymerase Pol zeta, and provide a mutational signature for mutagenic Pol zeta-dependent activity. In the current study, we have found that a specific subset of the Pol zeta-dependent mutational events requires oxidative metabolism. These results suggest that translesion bypass of spontaneously oxidized DNA bases can be a significant source of mutagenesis in repair compromised cells.