May 15, 2003 existing evidence indicates that set2, the histone 3 lysine 36 methyltransferase of saccharomyces cerevisiae, is a transcriptional repressor. The uses of genomewide yeast mutant collections genome. These drugs reduce intracellular nucleotide pools, which is thought to increase polymerase pausing, making transcription more. Because sit4 was first identified through mutations that affect rnapii transcription of a variety of genes a rndt et al. Zinc oxide and silver nanoparticles toxicity in the bakers. Cells were grown at 30 c in synthetic complete sc medium with appropriate amino acids and bases. Using this technique, we investigated the sensitivity of free and attached yeast cells to the lytic effect of 5bromo 6 azauracil against the compositional changes in wall polysaccharides and mannoproteins, stimulated in saccharomyces cerevisiae, candida utilis, and kluyveromyces lactis cells by their anchorage. It is a target of therapeutically useful drugs and is implicated in the regulation of cell growth rate. Functional profiling of the technical toxaphene organochlorinated pesticide mixture in yeast. B, immunoblot analysis of hybrid sii molecules expressed in tny14. Colonies were counted after 72 h of incubation at 30c. Repeatspecific functions for the cterminal domain of rna. Largescale screening of yeast mutants for sensitivity to. Characterization of sua7 mutations defines a domain of tfiib involved in transcription start site selection in yeast.
Sensitivity to 6au was tested by plating strains harboring prs316 51 onto plates lacking uracil and containing 25 g of 6au per ml. Mutants were chosen for analysis based on their ability to render yeast cells sensitive to growth on medium containing 6 azauracil. Mutations in the second largest subunit of rna polymerase. Strains without plasmid were tested for sensitivity to 1. Rpb2, rpb1, tfiis, and elp1 elicit 6azauracil 6au correlated with defects in. Such inhibitors include mycophenolate, 6azauracil 6au, tiazofurin, and. Regulation of histone h3k4 trimethylation and paf complex. The fidelity of transcription journal of biological chemistry. Novel rna polymerase ii mutation suppresses transcriptional.
Apr 01, 2006 rna polymerase ii rnapii in eukaryotic cells drives transcription of most messenger rnas. Largescale screening of yeast mutants for sensitivity to the imp dehydrogenase inhibitor 6 azauracil linda riles, 1 randal j. Mutations in the saccharomyces cerevisiae rpb1 gene. Largescale screening of yeast mutants for sensitivity to the. Threefold serial dilutions of indicated strains were spotted onto agar plates with or without each drug, starting at 1. Genomewide recruitment analysis of rpb4, a subunit of. Yeast cells lacking the elp2 gene display typical elp phenotypes including temperature, salt, and 6 azauracil sensitivity, and slow adaptation to growth on carbon sources such as galactose fellows et al. Methylation of histone h3 by set2 in saccharomyces cerevisiae.
Indeed, sensitivity to 6au was the first phenotype identified for cells with defective rna polymerase ii elongation factors, and it has been widely considered to be diagnostic for. Rna polymerase ii purified from three different 6azauracilsensitive yeast. This procedure has been successfully applied to detect c. Use of rna yeast polymerase ii mutants in studying. The extent of this defect did not correlate with sensitivity to growth in the presence of 6 azauracil.
Spt4 spt4spt5dsif complex is involved in rnapii promoterproximal pausing and it also has a positive role in transcription elongation as shown by 6azauracil 6au sensitivity. The taptagged rpb3 and rpb4 strains were cultured in 100 ml yeast extractpeptonedextrose medium at 25c until log phase optical density at 600 nm od 600of0. Spot testing yeast strains useful for testing uv, sensitivity to different media etc 1. Graphs show occupancy of pol ii subunit rpb3 in wt ysb2716 and rpb1e1103g ysb2717 strains containing a gal1ylr454 construct, before 0 min and after glucose addition 2, 4, 8 min to shut off gal1 promoterdriven transcription. Treatment of yeast with 6azauracil 6au leads to a reduction of intracellular gtp levels. In a study of zymocins mode of action, genetic scenarios known to impair transcription or affect the pol ii machinery itself were found to elicit hypersensitivity to zymocin. May 01, 2018, 1996 mutations in the second largest subunit of rna polymerase ii cause 6azauracil sensitivity in yeast and increased transcriptional arrest in vitro. Chip assays were performed essentially as described earlier 23. Shaw, 2 mark johnston, 1 and daniel reines 2, 1 department of genetics, washington university medical school, st.
Spotting assay spotting assay was performed as previously described song and ahn 2010. Yeast strains and growth conditionsstrains used in this study are listed in supplemental table 1. Rnapii core enzyme is composed of 12 polypeptides where rpb1 is the largest subunit. In characterization yeastrnapolymerase ii with reduced. To validate the glro assay, we measured transcription elongation in null mutants of genes with a known role in transcription elongation like spt4 and rpb9.
For the 6 azauracil sensitivity assay, each strain was transformed with the prs316 ura3 empty vector, spotted onto synthetic complete plates lacking uracil and leucine, and supplemented with 4 mgml 6 azauracil sigma. Addition of6auto ayeast culture results in a significant depletion of intracellular gtpand utppools 15. One of the limitations of the 6ausensitivity assay is that cells must be tested on uracilfree media since uracil competes with and neutralizes the. The elp2 subunit is essential for elongator complex assembly. A novel assay identifies transcript elongation roles for the.
Since reducing ntp concentrations in vitro decreases the elongation rate and increasespauseandarrest ofrnapolymerase1619,it has been hypothesized 15, 20 that a similar effect may be. Therefore, it is intriguing to assess the effects of 6 au. Most of the strains produced for this study were derived from the common laboratory strains w3031a mw644 and w3031b mw647 32. Jun 25, 2004 riles l, shaw rj, johnston m, reines d. Here, 10 or 5fold serial dilutions of the indicated strains were spotted on scu. Here we show by five main lines of evidence that set2 is involved in transcriptional elongation. Because rpb210 pol ii exhibited the most severe defect in these assays, this.
Selective and sensitive probe based in oligonucleotidecapped. The highly conserved, coregulated sno and snz gene families. This chapter discusses the application of 6azauracil 6au and mycophenolic acid mpa sensitivity as a genetic assay and will provide procedures for the purification of rna polymerase ii for use in in vitro transcription assays. Saccharomyces cerevisiae transcription elongation mutants are. Mutations in the second largest subunit of rna polymerase ii cause 6 azauracil sensitivity in yeast and increased transcriptional arrest in vitro.
Assay for yeast treatment of yeast with 6azauracil 6au leads to a reduction of intracellular gtp levels. For each liquidculture experiment, yeast cells were shaken at 250 rpm in 100 ml of medium at 30c for the time indicated see results. Pdf mutations in the second largest subunit of rna. Determining the toxaphene ic20 for functional profiling. For the 6au sensitivity assay, strains were grown in yeast extractpeptonedextrose medium for 24 h and diluted to an od 600 of 1. To further understand the mechanisms of rnapii transcription, we isolated and characterized novel point mutants of rpb1 that are sensitive to the nucleotidedepleting drug 6 azauracil 6au. Largescale screening of yeast mutants for sensitivity to the imp dehydrogenase inhibitor 6 azauracil. Request pdf 6azauracil sensitivity assay for yeast introductiontreatment of yeast with 6azauracil 6au leads to a reduction of intracellular gtp levels.
The reduction in gtp levels is not itself lethal, but can block yeast growth when combined with mutations that affect transcriptional elongation. In vivo elongation assay cells transformed with the gal1lacz plasmid were grown in scu medium containing 2% raffinose overnight, collected and subsequently shifted to. For mpa and 6azauracil sensitivity assays, yeast cells of 0. Strains transformed with the ura3 plasmid, prs316, were tested for sensitivity to 150 lgml of 6 au or 15 lgml of mpa.
Mutations in the second largest subunit of rna polymerase ii. Assay for yeast treatment of yeast with 6 azauracil 6au leads to a reduction of intracellular gtp levels. First, most, if not all, subunits of the rnap ii holoenzyme co. For the 6au sensitivity assay, cells were transformed with prs316. Suppression of 6azauracil sensitivity of yeast sii null mutants by sii hybrids. The mic 100 values of znonps and agnps using the yeast ypdagar assay were 1. The yeast escrt complexes are involved in the regulation of.
Mutations in the second largest subunit of rna polymerase ii cause 6azauracil sensitivity in yeast and increased transcriptional arrest in vitro. Confirming dieldrin sensitivity for various strains by flow cytometry. Yeast rna polymerase ii enzymes containing single amino acid substitutions in the second largest subunit were analyzed in vitro for elongationrelated defects. Mar 22, 1996 rna polymerase ii purified from three different 6 azauracil sensitive yeast strains displayed increased arrest at well characterized arrest sites in vitro. Yeast strains were grown at 30c to an optical density at 595 nm od 595of0. Rad26, the transcriptioncoupled repair factor in yeast, is. For chromatin immunoprecipitation chip experiments, all yeast strains were grown at 30 c to an a 600 of0. Use of rna yeast polymerase ii mutants in studying transcription. Identification of the region in yeast sii that defines. Yeast elongation mutants sensitive to 6au, such as those with a disrupted gene. Sit4p protein phosphatase is required for sensitivity of.
In the yeast saccharomyces cerevisiae, mutations in components of the rna polymerase ii pol ii transcription elongation machinery confer increased sensitivity to a drug that inhibits impdh, 6 azauracil 6au, by a mechanism that. This chapter discusses the application of 6 azauracil 6au and mycophenolic acid mpa sensitivity as a genetic assay and will provide procedures for the purification of rna polymerase ii for use in in vitro transcription assays. Feb 01, 2010 read novel rna polymerase ii mutation suppresses transcriptional fidelity and oxidative stress sensitivity in rpb9. Sep 12, 2002 the g1 cell cycle arrest imposed by kluyveromyces lactis zymocin on saccharomyces cerevisiae requires a functional rna polymerase ii pol ii totelongator complex.
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