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Mediator, a central coregulator of transcription, has been identified as a large protein complex in eukaryotes ranging from yeast to man. It is therefore remarkable that Mediator has not yet been identified within the plant kingdom. Here we identify Mediator in a plant, Arabidopsis thaliana. The plant Mediator subunits typically show very low homology to other species, but our biochemical purification identifies 21 conserved and six A. thaliana-specific Mediator subunits. Most notably, we identify the A. thaliana proteins STRUWWELPETER (SWP) and PHYTOCHROME AND FLOWERING TIME 1 (PFT1) as the Med14 and Med25 subunits, respectively. These findings show that specific plant Mediator subunits are linked to the regulation of specialized processes such as the control of cell proliferation and the regulation of flowering time in response to light quality. The identification of the plant Mediator will provide new tools and insights into the regulation of transcription in plants.

Sin4 and Rgrl proteins, previously shown by genetic studies to play both positive and negative roles in the transcriptional regulation of many genes, are identified here as components of mediator and RNA polymerase II holoenzyme complexes. Results with Sin4 deletion and Rgrl truncation strains indicate the association of these proteins in a subcomplex comprising Sin4, Rgrl, Galll, and a 50-kDa polypeptide. Taken together with the previous genetic evidence, our findings point to a role of the mediator in repression as well as in transcriptional activation. tory effects are sometimes seen at the same promoter. Sin4, like Rgrl, is required for repression of glucose-regulated genes, and both proteins are required for maximal induction of these genes as well (13).Here we report biochemical connections that may underlie the genetic similarities GAl1, SIN4, and RGR1. Our findings further indicate how the paradox of both positive and negative control by Galll, Sin4, and Rgrl proteins may be resolved. Finally, there are implications for the structure and role of the mediator complex in transcriptional regulation.

Development in plants is controlled by abiotic environmental cues such as day length, light quality, temperature, drought, and salinity. These signals are sensed by a variety of systems and transmitted by different signal transduction pathways. Ultimately, these pathways are integrated to control expression of specific target genes, which encode proteins that regulate development and differentiation. The molecular mechanisms for such integration have remained elusive. We here show that a linear 130-aminoacids-long sequence in the Med25 subunit of the Arabidopsis thaliana Mediator is a common target for the drought response element binding protein 2A, zinc finger homeodomain 1, and Myb-like transcription factors which are involved in different stress response pathways. In addition, our results show that Med25 together with drought response element binding protein 2A also function in repression of PhyB-mediated light signaling and thus integrate signals from different regulatory pathways.

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Ribonucleotide reductase in mammalian cells is composed of two nonidentical subunits, proteins R1 and R2, each inactive alone. The R1 protein is present in excess in proliferating cells, and its levels are constant during the cell cycle. Expression of the R2 protein, which is limiting for enzyme activity, is strictly S-phase-correlated. In this paper, we have used antisense RNA probes in a solution hybridization assay to measure the levels of R1 and R2 mRNA during the cell cycle in centrifugally elutriated cells and in cells synchronized by isoleucine or serum starvation. The levels of both transcripts were very low or undetectable in G0/G1-phase cells, showed a pronounced increase as cells progressed into S phase, and then declined when cells progressed into G2 + M phase. The R1 and R2 transcripts increased in parallel, starting slightly before the rise in S-phase cells, and reached the same levels. The relative lack of cell cycle dependent variation in R1 protein levels, obtained previously, may therefore simply be a consequence of the long half-life of the R1 protein. Hydroxyurea-resistant, R2-overproducing mouse TA3 cells showed the same regulation of the R1 and R2 transcripts as the parental cells, but with R2 mRNA at a 40-fold higher level.

Although the mechanisms of transcriptional regulation by RNA polymerase II are apparently highly conserved from yeast to man, the identification of a yeast TATA- The regulation of transcription of mRNA-encoding eukaryotic genes is a complicated process involving the modulation of chromatin structure, activities of upstream activators and repressors, and the concerted action of multiple components of the basal transcription machinery, including RNA polymerase It itself (1, 2). It is thought that the interaction of the TATA-binding protein (TBP), with the TATA-box promoter element is the first step in the formation of the RNA polymerase II preinitiation complex (PIC), and numerous studies have shown that PIC formation is subject to modulation by a variety of transcriptional regulators. However, the mechanisms by which these factors exert their effects are not yet fully understood. In metazoan systems, one basal factor that has been shown to be directly involved in mediating activation by upstream activators is the transcription factor TFIID, which is composed of TBP and TBP-associated factors (TAF11s