Mediator exists in a free form containing the Med12, Med13, CDK8, and CycC subunits (the Srb8-11 module) and a smaller form, which lacks these four subunits and associates with RNA polymerase II (Pol II), forming a holoenzyme. We use chromatin immunoprecipitation (ChIP) and DNA microarrays to investigate genome-wide localization of Mediator and the Srb8-11 module in fission yeast. Mediator and the Srb8-11 module display similar binding patterns, and interactions with promoters and upstream activating sequences correlate with increased transcription activity. Unexpectedly, Mediator also interacts with the downstream coding region of many genes. These interactions display a negative bias for positions closer to the 5' ends of open reading frames (ORFs) and appear functionally important, because downregulation of transcription in a temperature-sensitive med17 mutant strain correlates with increased Mediator occupancy in the coding region. We propose that Mediator coordinates transcription initiation with transcriptional events in the coding region of eukaryotic genes.

The human mitochondrial transcription termination factor (mTERF) is involved in the regulation of transcription of the mitochondrial genome. Similarity searches and phylogenetic analysis demonstrate that mTERF is a member of large and complex protein family (the MTERF family) shared amongst metazoans and plants. Interestingly, we identify three novel MTERF genes in vertebrates, which all encode proteins with predicted mitochondrial localization. Members of the MTERF family have so far not been detected in fungi, supporting the notion that mitochondrial transcription regulation may have evolved separately in yeast and animal cells.

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Mediator is an evolutionary conserved coregulator complex required for transcription of almost all RNA polymerase II-dependent genes. The Schizosaccharomyces pombe Mediator consists of two dissociable components—a core complex organized into a head and middle domain as well as the Cdk8 regulatory subcomplex. In this work we describe a functional characterization of the S. pombe Mediator. We report the identification of the S. pombe Med20 head subunit and the isolation of ts alleles of the core head subunit encoding med17+. Biochemical analysis of med8ts, med17ts, Δmed18, Δmed20 and Δmed27 alleles revealed a stepwise head domain molecular architecture. Phenotypical analysis of Cdk8 and head module alleles including expression profiling classified the Mediator mutant alleles into one of two groups. Cdk8 module mutants flocculate due to overexpression of adhesive cell-surface proteins. Head domain-associated mutants display a hyphal growth phenotype due to defective expression of factors required for cell separation regulated by transcription factor Ace2. Comparison with Saccharomyces cerevisiae Mediator expression data reveals that these functionally distinct modules are conserved between S. pombe and S. cerevisiae.

Edible microbial biomass derived from bacteria, yeasts, filamentous fungi or microalgae is a promising alternative to conventional sources of food and feed. Microorganisms are a good source of protein, vitamins and, in some cases, also contain beneficial lipids. The ability of microorganisms to use simple organic substrates for growth permits industrial-scale cultivation of edible microbial biomass in geographical locations that would not compete with agricultural production. Only a handful of microbial products are currently available for human consumption. The use of microbial biomass for animal feed is limited by access to low-cost growth substrates and competition from conventional feed sources such as soy and fishmeal. At a time when the global food production system is threatened by the effects of climate change, the production of edible microorganisms has the potential to circumvent many of the current environmental boundaries of food production as well as reducing its environmental impact. Photosynthetic microorganisms such as cyanobacteria and microalgae can be cultivated for food and feed independently of arable land. In addition, recent technological developments in atmospheric carbon dioxide (CO 2) capture, extraction and catalytic conversion into simple organic compounds can be used for cultivation of edible microbial biomass for food and feed in a manner that is wholly independent of photosynthesis. The future possibilities, challenges and risks of scaled-up production of edible microbial biomass in relation to the global food system and the environment are discussed.

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The fission yeast Schizosaccharomyces pombe has proved an important model system for cross-species comparative studies of many fundamental processes in the eukaryotic cell, such as cell cycle control and DNA replication. The RNA polymerase II transcription machinery is, however, still relatively poorly understood in S. pombe, partially due to the absence of a reconstituted in vitro transcription system. We have now purified S. pombe RNA polymerase II and its general initiation factors TFIIB, TFIIF, TFIIE, and TFIIH to near homogeneity. These factors enable RNA polymerase II to initiate transcription from the S. pombe alcohol dehydrogenase promoter (adh1p) when combined with Saccharomyces cerevisiae TATA-binding protein. We use our reconstituted system to examine effects of Mediator on basal transcription in vitro. S. pombe Mediator exists in two distinct forms, a free form, which contains the spSrb8, spTrap240, spSrb10, and spSrb11 subunits, and a smaller form, which lacks these four subunits and associates with RNA polymerase II to form a holoenzyme. We find that spSrb8/spTrap240/spSrb10/spSrb11 containing Mediator repress basal transcription, whereas Mediator lacking these subunits has a stimulatory effect on transcription. Our findings thus demonstrate that the spSrb8/spTrap240/spSrb10/spSrb11 subcomplex governs the ability of Mediator to stimulate or repress basal transcription in vitro.

CMO1 encodes a putative choline monooxygenase and is required for the utilization of choline as the sole nitrogen source in the yeast Scheffersomyces stipitis (syn. Pichia stipitis) Sixteen yeasts with sequenced genomes belonging to the ascomycete subphyla Saccharomycotina and Taphrinomycotina were assayed for their ability to utilize a variety of primary, secondary, tertiary and quartenary aliphatic amines as nitrogen sources. The results support a previously proposed pathway of quaternary amine catabolism whereby glycine betaine is first converted into choline, which is then cleaved to release trimethylamine, followed by stepwise demethylation of trimethylamine to release free ammonia. There were only a few instances of utilization of N-methylated glycine species (sarcosine and N,N-dimethylglycine), which suggests that this pathway is not intact in any of the species tested. The ability to utilize choline as a sole nitrogen source correlated strongly with the presence of a putative Rieske nonhaem iron protein homologous to bacterial ring-hydroxylating oxygenases and plant choline monooxygenases. Deletion of the gene encoding the Rieske non-haem iron protein in the yeast Scheffersomyces stipitis abolished its ability to utilize choline as the sole nitrogen source, but did not affect its ability to use methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, ethanolamine or glycine as nitrogen sources. The gene was named CMO1 for putative choline monooxygenase 1. A bioinformatic survey of eukaryotic genomes showed that CMO1 homologues are found throughout the eukaryotic domain.