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Alternative Pol II transcription
headlines
Mass-spec spawns spate of spindle-pole parts
Alternative Pol II transcription
Wigge, P. A., Jensen, O. N., Holmes, S., Soues, S., Mann, M. and Kilmartin, J. V. (1998) Analysis of the Saccharomyces spindle pole by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry J. Cell Biol. 141, 967–977
LEE, D. and LIS, J. T. (1998) Transcriptional activation independent of TFIIH kinase and the RNA polymerase II mediator in vivo Nature 393, 389–392
The yeast spindle pole body (SPB) is the equivalent of the centrosome in higher eukaryotes and is essential for mitosis. A prerequisite for understanding SPB assembly and function is to identify its protein components. Wigge et al. purified yeast SPBs and applied matrixassisted laser desorption ionization (MALDI) mass spectrometry protein identification. This method works in two steps. First, MALDI mass spectrometry, a precise way to measure peptide mass, is used to find the exact masses of tryptic peptides. Then, the yeast database is used to predict the tryptic peptide masses for every ORF in the genome, which are matched to the actual masses to determine which gene encodes the protein, thus identifying both the protein and the gene. By running MALDI mass spectrometry on SDS–PAGE bands enriched in the SPB fraction, Wigge et al. identified 12 of the 17 previously known components of the SPB and spindle. They also found six previously identified proteins of unknown function or localization plus five completely new proteins. SPB localization of these 11 novel SPB components was verified by immunofluorescence using haemagglutinin (HA)-tagged proteins, visualization of green fluorescent protein (GFP) fusions and by immunoelectron microscopy. Wigge and colleagues knocked out all 11 genes – eight of the mutants were lethal, the other three grew slowly. One essential component, Ndc80p, is homologous to a human centromere-localized protein, and temperature-sensitive alleles of NDC80 gave a chromosome-segregation defect. This work not only reveals new SPB components for future study but also demonstrates the power of MALDI mass spectrometry for identifying the protein components of yeast complexes.
The mechanism of transcriptional activation is still poorly understood, as illustrated by experiments carried out by Lee and Lis. KIN28 is an essential gene encoding a protein kinase subunit of the general transcription factor TFIIH in yeast. The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) becomes multiply phosphorylated subsequent to interaction with the promoter and prior to initiation of gene transcription. Kin28p has been thought to be the kinase responsible because, for all genes tested, transcription is inhibited at the nonpermissive temperature in temperature-sensitive kin28 mutants. However, Lee and Lis have now shown that CUP1, a copper-inducible gene and SSA4, the heat-shock protein 70 gene, both remain highly inducible by copper sulphate and heat-shock, respectively, in a conditional kin28 mutant. The CUP1 and SSA4 genes were activated normally in two other strains, each lacking a putative CTD kinase. Therefore, at least three known yeast CTD kinases are dispensable for activation of CUP1 and SSA4, indicating that phosphorylation might not be necessary for transcription of these genes. One model of transcriptional activation proposes that upstream activators interact with a CTD-bound mediator complex to recruit the holoenzyme form of Pol II to the promoter. CTD phosphorylation might be a way of allowing promoter clearance after transcription has been initiated as the phosphorylated CTD does not interact with the mediator complex. Using temperature-sensitive mutants of Srb4p and Srb6p, two crucial subunits of the mediator complex, Lee and Lis demonstrate that Kin28p-dependent transcription is also mediator dependent. These data are in agreement with the proposed model. By contrast, CUP1 and SSA4 gene activation is induced to comparably high levels in Kin28p-, Srb4p- and Srb6p-depleted mutants. From these findings, it must be concluded that some genes require neither TFIIH kinase nor crucial components of the Pol II mediator complex for activated transcription.
cytobytes http://www.cellbio.com/ This is a cell and molecular biology jump station maintained by Pamela Gannon. There are links to a whole host of Web sites that are relevant to molecular and cell biologists, including electronic publications, methods and protocols, educational resources, careers resources, professional societies and conferences. The author makes a specific effort to highlight only sites that are particularly useful and well-organized, and not surprisingly her own site is too!
trends in CELL BIOLOGY (Vol. 8) August 1998
309
Mass-spec spawns spate of spindle-pole parts
Alternative Pol II transcription
Wigge, P. A., Jensen, O. N., Holmes, S., Soues, S., Mann, M. and Kilmartin, J. V. (1998) Analysis of the Saccharomyces spindle pole by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry J. Cell Biol. 141, 967–977
LEE, D. and LIS, J. T. (1998) Transcriptional activation independent of TFIIH kinase and the RNA polymerase II mediator in vivo Nature 393, 389–392
The yeast spindle pole body (SPB) is the equivalent of the centrosome in higher eukaryotes and is essential for mitosis. A prerequisite for understanding SPB assembly and function is to identify its protein components. Wigge et al. purified yeast SPBs and applied matrixassisted laser desorption ionization (MALDI) mass spectrometry protein identification. This method works in two steps. First, MALDI mass spectrometry, a precise way to measure peptide mass, is used to find the exact masses of tryptic peptides. Then, the yeast database is used to predict the tryptic peptide masses for every ORF in the genome, which are matched to the actual masses to determine which gene encodes the protein, thus identifying both the protein and the gene. By running MALDI mass spectrometry on SDS–PAGE bands enriched in the SPB fraction, Wigge et al. identified 12 of the 17 previously known components of the SPB and spindle. They also found six previously identified proteins of unknown function or localization plus five completely new proteins. SPB localization of these 11 novel SPB components was verified by immunofluorescence using haemagglutinin (HA)-tagged proteins, visualization of green fluorescent protein (GFP) fusions and by immunoelectron microscopy. Wigge and colleagues knocked out all 11 genes – eight of the mutants were lethal, the other three grew slowly. One essential component, Ndc80p, is homologous to a human centromere-localized protein, and temperature-sensitive alleles of NDC80 gave a chromosome-segregation defect. This work not only reveals new SPB components for future study but also demonstrates the power of MALDI mass spectrometry for identifying the protein components of yeast complexes.
The mechanism of transcriptional activation is still poorly understood, as illustrated by experiments carried out by Lee and Lis. KIN28 is an essential gene encoding a protein kinase subunit of the general transcription factor TFIIH in yeast. The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) becomes multiply phosphorylated subsequent to interaction with the promoter and prior to initiation of gene transcription. Kin28p has been thought to be the kinase responsible because, for all genes tested, transcription is inhibited at the nonpermissive temperature in temperature-sensitive kin28 mutants. However, Lee and Lis have now shown that CUP1, a copper-inducible gene and SSA4, the heat-shock protein 70 gene, both remain highly inducible by copper sulphate and heat-shock, respectively, in a conditional kin28 mutant. The CUP1 and SSA4 genes were activated normally in two other strains, each lacking a putative CTD kinase. Therefore, at least three known yeast CTD kinases are dispensable for activation of CUP1 and SSA4, indicating that phosphorylation might not be necessary for transcription of these genes. One model of transcriptional activation proposes that upstream activators interact with a CTD-bound mediator complex to recruit the holoenzyme form of Pol II to the promoter. CTD phosphorylation might be a way of allowing promoter clearance after transcription has been initiated as the phosphorylated CTD does not interact with the mediator complex. Using temperature-sensitive mutants of Srb4p and Srb6p, two crucial subunits of the mediator complex, Lee and Lis demonstrate that Kin28p-dependent transcription is also mediator dependent. These data are in agreement with the proposed model. By contrast, CUP1 and SSA4 gene activation is induced to comparably high levels in Kin28p-, Srb4p- and Srb6p-depleted mutants. From these findings, it must be concluded that some genes require neither TFIIH kinase nor crucial components of the Pol II mediator complex for activated transcription.
cytobytes http://www.cellbio.com/ This is a cell and molecular biology jump station maintained by Pamela Gannon. There are links to a whole host of Web sites that are relevant to molecular and cell biologists, including electronic publications, methods and protocols, educational resources, careers resources, professional societies and conferences. The author makes a specific effort to highlight only sites that are particularly useful and well-organized, and not surprisingly her own site is too!
trends in CELL BIOLOGY (Vol. 8) August 1998
309