The TFE-induced transient native-like structure of the intrinsically disordered σ47° domain of Escherichia coli RNA polymerase.

The transient folding of domain 4 of an E. coli RNA polymerase σ7° subunit (rECσ47°) induced by an increasing concentration of 2,2,2-trifluoroethanol (TFE) in an aqueous solution was monitored by means of CD and heteronuclear NMR spectroscopy. NMR data, collected at a 30% TFE, allowed the estimation of the population of a locally folded rECσ47° structure (CSI descriptors) and of local backbone dynamics ((15)N relaxation). The spontaneous organization of the helical regions of the initially unfolded protein into a TFE-induced 3D structure was revealed from structural constraints deduced from (15)N- to (13)C-edited NOESY spectra. In accordance with all the applied criteria, three highly populated α-helical regions, separated by much more flexible fragments, form a transient HLHTH motif resembling those found in PDB structures resolved for homologous proteins. All the data taken together demonstrate that TFE induces a transient native-like structure in the intrinsically disordered protein.

Combined in silico and 19F NMR analysis of 5-fluorouracil metabolism in yeast at low ATP conditions.

The cytotoxic effect of 5-fluorouracil (5-FU) on yeast cells is thought to be mainly via a misincorporation of fluoropyrimidines into both RNA and DNA, not only DNA damage via inhibition of thymidylate synthase (TYMS) by fluorodeoxyuridine monophosphate (FdUMP). However, some studies on Saccharomyces cerevisiae show a drastic decrease in ATP concentration under oxidative stress, together with a decrease in concentration of other tri- and diphosphates. This raises a question if hydrolysis of 5-fluoro-2-deoxyuridine diphosphate (FdUDP) under oxidative stress could not lead to the presence of FdUMP and the activation of so-called 'thymine-less death' route. We attempted to answer this question with in silico modeling of 5-FU metabolic pathways, based on new experimental results, where the stages of intracellular metabolism of 5-FU in Saccharomyces cerevisiae were tracked by a combination of 19F and 31P NMR spectroscopic study. We have identified 5-FU, its nucleosides and nucleotides, and subsequent di- and/or triphosphates. Additionally, another wide 19F signal, assigned to fluorinated unstructured short RNA, has been also identified in the spectra. The concentration of individual metabolites was found to vary substantially within hours, however, the initial steady-state was preserved only for an hour, until the ATP concentration dropped by a half, which was monitored independently via 31P NMR spectra. After that, the catabolic process leading from triphosphates through monophosphates and nucleosides back to 5-FU was observed. These results imply careful design and interpretation of studies in 5-FU metabolism in yeast.

Regulation of sporulation in the yeast Saccharomyces cerevisiae.

Sporulation of the budding yeast Saccharomyces cerevisiae ­ equivalent to gametogenesis in higher organisms, is a complex differentiation program induced by starvation of cells for nitrogen and carbon. Such environmental conditions activate coordinated, sequential changes in gene expression leading to production of haploid, stress-resistant spores. Sporulation comprises two rounds of meiosis coupled with spore morphogenesis and is tightly controlled to ensure viable progeny. This review concerns the regulation of differentiation process by nutritional and transcriptional signals.

Mutants of the Saccharomyces cerevisiae VPS genes CCZ1 and YPT7 are blocked in different stages of sporulation.

The CCZ1 gene is a member of the class B VPS (vacuolar protein sorting) genes and it is engaged in the last stage of delivery of multiple kinds of cargo to the yeast vacuole. In the process of fusion of the multivesicular body (MVB) with the vacuole, Ccz1p forms a complex with Ypt7p. Both genes are non-essential for vegetative growth, but their deletions cause a complete block in spore formation. The results of this study indicate that ccz1Δ cells initiate the meiotic program, properly proceed through premeiotic DNA replication and through the pairing of homologous chromosomes, but fail to progress through the first meiotic divisions and arrest in prophase I with a single nucleus. The mutant cells are defective in spindle formation as well as in duplication and/or separation of the SPBs. ypt7Δ cells, on the other hand, cannot execute DNA synthesis. We also show that expression of a mutated variant of the YPT7 gene suppresses the sporulation and autophagy defects of ccz1Δ cells to a quantitatively similar level, suggesting that restoration of autophagy in the ccz1Δ strain is sufficient to enable its sporulation.

Fcf1p and Fcf2p are novel nucleolar Saccharomyces cerevisiae proteins involved in pre-rRNA processing.

The uncharacterized Saccharomyces cerevisiae proteins Fcf1 and Fcf2, encoded by the ORFs YDR339c and YLR051c, respectively, were identified in a tandem affinity purification experiment of the known 40S factor Faf1p. Most of the proteins associated with TAP-Faf1p are trans-acting factors involved in pre-rRNA processing and 40S subunit biogenesis, in agreement with the previously observed role of Faf1p in 18S rRNA synthesis. Fcf1p and Fcf2p are both essential and localize to the nucleolus. Depletion of Fcf1p and Fcf2p leads to a decrease in synthesis of the 18S rRNA, resulting in a deficit in 40S ribosomal subunits. Northern analysis indicates inefficient processing of pre-rRNA at the A(0), A(1), and A(2) cleavage sites.

Functional and physical interactions of Krr1p, a Saccharomyces cerevisiae nucleolar protein.

The Krr1 protein of Saccharomyces cerevisiae is involved in processing of pre-rRNA and assembly of pre-ribosomal 40S subunits. To further investigate the function of Krr1p we constructed a conditional cold sensitive mutant krr1-21, and isolated seven genes from Schizosaccharomyces pombe whose products suppressed the cold sensitive phenotype of krr1-21 cells. Among the multicopy suppressors we found genes coding for translation elongation factor EF-1alpha, a putative ribose methyltransferase and five genes encoding ribosomal proteins. Using the tandem affinity purification (TAP) method we identified thirteen S. cerevisiae ribosomal proteins interacting with Krr1p. Taken together, these results indicate that Krr1p interacts functionally as well as physically with ribosomal proteins. Northern blot analysis revealed that changes in the level of krr1-21 mRNA were accompanied by similar changes in the level of mRNAs of genes encoding ribosomal proteins. Thus, Krr1p and the genes encoding ribosomal proteins it interacts with seem to be coordinately regulated at the level of transcription.

Functional and physical interactions of Faf1p, a Saccharomyces cerevisiae nucleolar protein.

We report the discovery and characterisation of a novel nucleolar protein of Saccharomyces cerevisiae. We identified this protein encoded by ORF YIL019w, designated in SGD base as Faf1p, in a two hybrid interaction screen using the known nucleolar protein Krr1 as bait. The presented data indicate that depletion of the Faf1 protein has an impact on the 40S ribosomal subunit biogenesis resulting from a decrease in the production of 18S rRNA. The primary defect is apparently due to inefficient processing of 35S rRNA at the A(0), A(1), and A(2) cleavage sites.