Abstract
Med6 protein (Med6p) is a hallmark component of evolutionarily conserved Mediator complexes, and the genuine role of Med6p in Mediator functions remains elusive. For the functional analysis of Saccharomyces cerevisiae Med6p (scMed6p), we generated a series of scMed6p mutants harboring a small internal deletion. Genetic analysis of these mutants revealed that three regions (amino acids 33–42 (Δ2), 125–134 (Δ5), and 157–166 (Δ6)) of scMed6p are required for cell viability and are located at highly conserved regions of Med6 homologs. Notably, the Med6p-Δ2 mutant was barely detectable in whole-cell extracts and purified Mediator, suggesting a loss of Mediator association and concurrent rapid degradation. Consistent with this, the recombinant forms of Med6p having these mutations partially (Δ2) restore or fail (Δ5 and Δ6) to restore in vitro transcriptional defects caused by temperature-sensitive med6 mutation. In an artificial recruitment assay, Mediator containing a LexA-fused wild-type Med6p or Med6p-Δ5 was recruited to the lexA operator region with TBP and activated reporter gene expression. However, the recruitment of Mediator containing LexA-Med6p-Δ6 to lexA operator region resulted in neither TBP recruitment nor reporter gene expression. This result demonstrates a pivotal role of Med6p in the postrecruitment function of Mediator, which is essential for transcriptional activation by Mediator.
Highlights
Promoter-specific mRNA synthesis requires a minimal set of proteins comprising Pol RNA polymerase II ts (II) (RNA polymerase II) and associated GTFs, which is defined as in vitro “basal transcription” [1]
We dissected the functional domains of yeast Med6 protein (Med6p) on the basis of its requirement for cell viability and transcriptional activation via molecular genetics and biochemical approaches. e functional domains of Saccharomyces cerevisiae Med6p (scMed6p) (Δ2, Δ5, and Δ6 regions) identified by complementation assays were mapped to the highly conserved regions of Med6ps, whereas yeast-specific and mostly divergent regions of scMed6p were not required for its essential function(s) (Figures 1 and 2). ree lethal deletion mutants had defects in transcriptional activation in a reconstituted in vitro system only the Gal4-VP16 activator was tested in our in vitro transcription (Figure 4). ese results again clearly demonstrate that the major and essential function of Med6p is focused on transcriptional activation
In contrast to Med6p-Δ5 and -Δ6 mutants, the Med6p-Δ2 protein was barely detectable in whole-cell extracts (WCEs) and was even absent in enriched Pol II-Mediator fraction
Summary
Promoter-specific mRNA synthesis requires a minimal set of proteins comprising Pol II (RNA polymerase II) and associated GTFs (general transcription factors), which is defined as in vitro “basal transcription” [1] Regulated responses of this minimal transcription system by DNA-binding transcription factors or in vitro transcription from nucleosomal DNA template require additional sets of coregulator proteins involved in chromatin remodeling/modification as well as targeted recruitment of basal transcription machinery to the promoter [2, 3]. In vivo, these transcriptional cofactors participate in the regulated expression of specific genes in response to environmental and physiological cues, which has been demonstrated in a wide range of eukaryotic model organisms. A subset of Srb/Ssn proteins was identified as the distinct kinase module whose association with core Mediator is known to suppress the positive function of Mediator [9, 10]
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