Electron crystal structure of an RNA polymerase II transcription elongation complex.
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The structure of an actively transcribing complex, containing yeast RNA polymerase II with associated template DNA and product RNA, was determined by electron crystallography. Nucleic acid, in all likelihood the "transcription bubble" at the active center of the enzyme, occupies a previously noted 25 A channel in the protein structure. Details are indicative of a roughly 90 degrees bend of the DNA between upstream and downstream regions. The DNA apparently lies entirely on one face of the polymerase, rather than passing through a hole to the opposite side, as previously suggested. (+info)
The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif.
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The major pathway of mRNA degradation in yeast occurs through deadenylation, decapping and subsequent 5' to 3' exonucleolytic decay of the transcript body. To identify proteins that control the activity of the decapping enzyme, which is encoded by the DCP1 gene, we isolated a high-copy suppressor of the temperature-sensitive dcp1-2 allele, termed DCP2. Overexpression of Dcp2p partially suppressed the dcp1-2 decapping defect. Moreover, the Dcp2 protein was required for the decapping of both normal mRNAs and aberrant transcripts that are degraded by the mRNA surveillance pathway. The Dcp2 protein contains a MutT motif, which is found in a class of pyrophosphatases. Mutational analyses indicated that the region of Dcp2p containing the MutT motif is necessary and sufficient for Dcp2p's function in mRNA decapping. The Dcp2p also coimmunoprecipitates with the DCP1 decapping enzyme and is required for the production of enzymatically active decapping enzyme. These results suggest that direct or indirect interaction of Dcp1p with Dcp2p is required for the production of active decapping enzyme, perhaps in a process requiring the hydrolysis of a pyrophosphate bond. (+info)
An RNA binding motif in the Cbp2 protein required for protein-stimulated RNA catalysis.
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The fifth and terminal intron of yeast cytochrome b pre-mRNA (a group I intron) requires a protein encoded by the nuclear gene CBP2 for splicing. Because catalysis is intrinsic to the RNA, the protein is believed to promote formation of secondary and tertiary structure of the RNA, resulting in a catalytically competent intron. In vitro, this mitochondrial intron can be made to self-splice or undergo protein-facilitated splicing by varying the Mg(2+) and monovalent salt concentrations. This two-component system, therefore, provides a good model for understanding the role of proteins in RNA folding. A UV cross-linking experiment was initiated to identify RNA binding sites on Cbp2 and gain insights into Cbp2-intron interactions. A 12-amino acid region containing a presumptive contact site near the amino terminus was targeted for mutagenesis, and mutant proteins were characterized for RNA binding and stimulation of splicing. Mutations in this region resulted in partial or complete loss of function, demonstrating the importance of this determinant for stimulation of RNA splicing. Several of the mutations that severely reduced splicing did not significantly shift the overall binding isotherm of Cbp2 for the precursor RNA, suggesting that contacts critical for activity are not necessarily reflected in the dissociation constant. This analysis has identified a unique RNA binding motif of alternating basic and aromatic residues that is essential for protein facilitated splicing. (+info)
The Saccharomyces cerevisiae MER3 gene, encoding a novel helicase-like protein, is required for crossover control in meiosis.
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The MER3 gene is identified as a novel meiosis-specific gene, whose transcript is spliced in an MRE2/MER1-dependent manner. The predicted Mer3 protein contains the seven motifs characteristic of the DExH-box type of helicases as well as a putative zinc finger. Double strand breaks (DSBs), the initial changes of DNA in meiotic recombination, do not disappear completely and are hyperresected late in mer3 meiosis, indicating that MER3 is required for the transition of DSBs to later intermediates. A mer3 mutation reduces crossover frequencies, and the remaining crossovers show random distribution along a chromosome, resulting in a high incidence of non-disjunction of homologous chromosomes at the first meiotic division. MER3 appears to be very important for both the DSB transition and crossover control. (+info)
Rat8p/Dbp5p is a shuttling transport factor that interacts with Rat7p/Nup159p and Gle1p and suppresses the mRNA export defect of xpo1-1 cells.
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In a screen for temperature-sensitive mutants of Saccharomyces cerevisiae defective for mRNA export, we previously identified the essential DEAD-box protein Dbp5p/Rat8p and the nucleoporin Rat7p/Nup159p. Both are essential for mRNA export. Here we report that Dbp5p and Rat7p interact through their Nterminal domains. Deletion of this portion of Rat7p (Rat7pDeltaN) results in strong defects in mRNA export and eliminates association of Dbp5p with nuclear pores. Overexpression of Dbp5p completely suppressed the growth and mRNA export defects of rat7DeltaN cells and resulted in weaker suppression in cells carrying rat7-1 or the rss1-37 allele of GLE1. Dbp5p interacts with Gle1p independently of the N-terminus of Dbp5p. Dbp5p shuttles between nucleus and cytoplasm in an Xpo1p-dependent manner. It accumulates in nuclei of xpo1-1 cells and in cells with mutations affecting Mex67p (mex67-5), Gsp1p (Ran) or Ran effectors. Overexpression of Dbp5p prevents nuclear accumulation of mRNA in xpo1-1 cells, but does not restore growth, suggesting that the RNA export defect of xpo1-1 cells may be indirect. In a screen for high-copy suppressors of the rat8-2 allele of DBP5, we identified YMR255w, now called GFD1. Gfd1p is not essential, interacts with Gle1p and Rip1p/Nup42p, and is found in the cytoplasm and at the nuclear rim. (+info)
Identification of Fusarium species involved in human infections by 28S rRNA gene sequencing.
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Fusarium spp. have emerged as major opportunistic fungal agents. Since new antifungal agents exhibit variable activity against Fusarium isolates depending on the species, rapid identification at the species level is required. Conventional culture methods are difficult, fastidious, and sometimes inconclusive. In this work, we sequenced a 440-bp fragment encoding the 28S rRNA from 33 Fusarium isolates belonging to six Fusarium species associated with human infections. The data were then analyzed by the neighbor-joining method. By using distance matrix analysis and constructing the phylogram, we could easily distinguish the different species for all but one isolate. The method also allowed differentiation between the closely related genera Acremonium and Cylindrocarpon. In contrast to the case with conventional methods, the results could be obtained within 48 h from a 3-day culture and are independent of mycologist experience, making this method rapid and reliable for identification of Fusarium species isolated from patients. (+info)
A sampling of the yeast proteome.
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In this study, we examined yeast proteins by two-dimensional (2D) gel electrophoresis and gathered quantitative information from about 1,400 spots. We found that there is an enormous range of protein abundance and, for identified spots, a good correlation between protein abundance, mRNA abundance, and codon bias. For each molecule of well-translated mRNA, there were about 4,000 molecules of protein. The relative abundance of proteins was measured in glucose and ethanol media. Protein turnover was examined and found to be insignificant for abundant proteins. Some phosphoproteins were identified. The behavior of proteins in differential centrifugation experiments was examined. Such experiments with 2D gels can give a global view of the yeast proteome. (+info)
Differential regulation of the cell wall integrity mitogen-activated protein kinase pathway in budding yeast by the protein tyrosine phosphatases Ptp2 and Ptp3.
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Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity and protein tyrosine phosphatases (PTPs) in yeasts. In Saccharomyces cerevisiae, two PTPs, Ptp2 and Ptp3, inactivate the MAPKs, Hog1 and Fus3, with different specificities. To further examine the functions and substrate specificities of Ptp2 and Ptp3, we tested whether they could inactivate a third MAPK, Mpk1, in the cell wall integrity pathway. In vivo and in vitro evidence indicates that both PTPs inactivate Mpk1, but Ptp2 is the more effective negative regulator. Multicopy expression of PTP2, but not PTP3, suppressed growth defects due to the MEK kinase mutation, BCK1-20, and the MEK mutation, MKK1-386, that hyperactivate this pathway. In addition, deletion of PTP2, but not PTP3, exacerbated growth defects due to MKK1-386. Other evidence supported a role for Ptp3 in this pathway. Expression of MKK1-386 was lethal in the ptp2Delta ptp3Delta strain but not in either single PTP deletion strain. In addition, the ptp2Delta ptp3Delta strain showed higher levels of heat stress-induced Mpk1-phosphotyrosine than the wild-type strain or strains lacking either PTP. The PTPs also showed differences in vitro. Ptp2 was more efficient than Ptp3 at binding and dephosphorylating Mpk1. Another factor that may contribute to the greater effectiveness of Ptp2 is its subcellular localization. Ptp2 is predominantly nuclear whereas Ptp3 is cytoplasmic, suggesting that active Mpk1 is present in the nucleus. Last, PTP2 but not PTP3 transcript increased in response to heat shock in a Mpk1-dependent manner, suggesting that Ptp2 acts in a negative feedback loop to inactivate Mpk1. (+info)