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Evidence that RNA polymerase II and not TFIIB is responsible for the difference in transcription initiation patterns between Saccharomyces cerevisiae and Schizosaccharomyces pombe.

ABSTRACT: The basal eukaryotic transcription machinery for protein coding genes is highly conserved from unicellular yeast to higher eukaryotes. Whereas TATA-containing promoters in human cells usually contain a single transcription start site (TSS) located ? 30 bp downstream of the TATA element, transcription in the yeast Schizosaccharomyces pombe and Saccharomyces cerevisiae typically initiates at multiple sites within a window ranging from 30 to 70 bp or 40 to 200 bp downstream of a TATA element, respectively. By exchanging highly purified factors between reconstituted S. pombe and S. cerevisiae transcription systems, we confirmed previous observations that the dual exchange of RNA polymerase II (RNAPII) and transcription factor IIB (TFIIB) confer the distinct initiation patterns between these yeast species. Surprisingly, however, further genetic and biochemical assays of TFIIB chimeras revealed that TFIIB and the proposed B-finger/reader domain do not play a role in determining the distinct initiation patterns between S. pombe and S. cerevisiae, but rather, these patterns are solely due to differences in RNAPII. These results are discussed within the context of a proposed model for the mechanistic coupling of the efficiency of early phosphodiester bond formation during productive TSS utilization and intrinsic elongation proficiency.

SUBMITTER: Yang C

PROVIDER: S-EPMC3413132 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Evidence that RNA polymerase II and not TFIIB is responsible for the difference in transcription initiation patterns between Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Yang Chen C Ponticelli Alfred S AS

Nucleic acids research 20120417 14

The basal eukaryotic transcription machinery for protein coding genes is highly conserved from unicellular yeast to higher eukaryotes. Whereas TATA-containing promoters in human cells usually contain a single transcription start site (TSS) located ∼ 30 bp downstream of the TATA element, transcription in the yeast Schizosaccharomyces pombe and Saccharomyces cerevisiae typically initiates at multiple sites within a window ranging from 30 to 70 bp or 40 to 200 bp downstream of a TATA element, respe ...[more]

PMID: 22510268

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Project description:The Pneumocystis genus is an opportunistic fungal pathogen that infects patients with AIDS and immunocompromised individuals. The study of this fungus has been hampered due to the inability to grow it in a (defined media/pure) culture. However, the use of modern molecular techniques and genomic analysis has helped researchers to understand its complex cell biology. The transcriptional process in the Pneumocystis genus has not been studied yet, although it is assumed that it has conventional transcriptional machinery. In this work, we have characterized the function of the RNA polymerase II (RNAPII) general transcription factor TFIIB from Pneumocystis carinii using the phylogenetically related biological model Schizosaccharomyces pombe. The results of this work show that Pneumocystis carinii TFIIB is able to replace the essential function of S. pombe TFIIB both in in vivo and in vitro assays. The S. pombe strain harboring the P carinii TFIIB grew slower than the parental wild-type S. pombe strain in complete media and in minimal media. The S. pombe cells carrying out the P. carinii TFIIB are larger than the wild-type cells, indicating that the TFIIB gene replacement confers a phenotype, most likely due to defects in transcription. P. carinii TFIIB forms very weak complexes with S. pombe TATA-binding protein on a TATA box promoter but it is able to form stable complexes in vitro when S. pombe TFIIF/RNAPII are added. P. carinii TFIIB can also replace the transcriptional function of S. pombe TFIIB in an in vitro assay. The transcription start sites (TSS) of the endogenous adh gene do not change when P. carinii TFIIB replaces S. pombe TFIIB, and neither does the TSS of the nmt1 gene, although this last gene is poorly transcribed in vivo in the presence of P. carinii TFIIB. Since transcription by RNA polymerase II in Pneumocystis is poorly understood, the results described in this study are promising and indicate that TFIIB from P. carinii can replace the transcriptional functions of S. pombe TFIIB, although the cells expressing the P. carini TFIIB show an altered phenotype. However, performing studies using a heterologous approach, like this one, could be relevant to understanding the basic molecular processes of Pneumocystis such as transcription and replication.

Project description:The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth. Herein, we report that S. pombe, spo20(+) is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle. We also demonstrate that the spo20(+) gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast. This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other. Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein. That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104 mutant accumulates aberrant Golgi cisternae at restrictive temperatures. However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation. Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells. We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus. This nuclear localization persists during conjugation and meiosis. On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells. In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus. Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function.

Dataset Information

Evidence that RNA polymerase II and not TFIIB is responsible for the difference in transcription initiation patterns between Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Publications

Evidence that RNA polymerase II and not TFIIB is responsible for the difference in transcription initiation patterns between Saccharomyces cerevisiae and Schizosaccharomyces pombe.

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