Structural and Functional Characterization of PC2 and RNA Polymerase II-Associated Subpopulations of Metazoan Mediator

Abstract

The coactivator complexes TRAP/SMCC and PC2 represent two forms of Mediator. To further understand the implications of the heterogeneity of the cellular Mediator populations for regulation of RNA polymerase II (Pol II) transcription, we used a combination of affinity and conventional chromatographic methods. Our analysis revealed a spectrum of complexes, including some containing significant proportions of Pol II. Interestingly, the subunit composition of the Pol II-associated Mediator population resembled that of PC2 more closely than that of the larger TRAP/SMCC complex. In in vitro transcription assays reconstituted from homogeneous preparations of general transcription factors, Mediator-associated Pol II displayed a greater specific activity (relative to that of standard Pol II) in activator-independent (basal) transcription in addition to the previously described effects of Mediator on activator-dependent transcription. Purified PC2 complex also stimulated basal activity under these conditions. Immobilized template assays in which activator-recruited preinitiation complexes were allowed to undergo one cycle of transcription revealed partial disruption of Mediator that resulted in a PC2-like complex being retained in the scaffold. This result implies that PC2 could originate as a result of a normal cellular process. Our results are thus consistent with a dynamic nature of the Mediator complex and further extend the functional similarities between Saccharomyces cerevisiae and metazoan Mediator complexes.

STAGA Recruits Mediator to the MYC Oncoprotein To Stimulate Transcription and Cell Proliferation

“…This model is in accord with previous reports indicating that MYC activates transcription of TERT and other target genes after Pol II engagement, possibly by stimulating Pol II C-terminal domain phosphorylation and/or promoter clearance or elongation (8,17,20) and with recent studies indicating a role of yeast SAGA in transcription elongation (12,27,76,78). Similarly, Mediator has also been shown to play a role postrecruitment of Pol II during the initiation phase of transcription in mouse cells (74), stimulates TFIIB recruitment and initiation/reinitiation in vitro (2,45,85), and physically and/or functionally associates with various elongation factors, including TFIIS (76), Brd4/P-TEFb (84), and DSIF (46), and with coding regions of yeast genes (1,87). Clearly, it will be important to identify the steps downstream of TFIID/Pol II loading that are controlled by STAGA and core Mediator on the TERT promoter and the detailed activation mechanisms involved.…”

Identification of a regulator of transcription elongation as an accessory factor for the human Mediator coactivator

“…The original system was reconstituted from homogeneous preparations of Pol II, GTFs, and PC4. Purified Mediator [either intact TRAP/SMCC (20,21) or PC2, the form that mainly lacks the TRAP240/MED13-TRAP230/MED12-CDK8-CycC subcomplex (13,22)] could be shown to potentiate both basal and activated transcription. Similarly, in a Mediator-depleted nuclear extract system, Mediator dependence of basal and activated transcription was readily demonstrable (10,12,14,17).…”

“…Importantly, in contrast to chromatin coactivators, Mediator can facilitate DNA-templated transcription, and thus acts relatively late in the overall activation pathway on natural templates (8). Several lines of evidence suggest that Mediator function is manifested both at the level of the initial establishment of the PIC as well as its postrecruitment function (5,7,(9)(10)(11)(12)(13)(14)(15).…”

“…Despite the critical requirement of Mediator for the expression of nearly all genes in yeast (16) and, potentially, metazoan cells, it has been puzzling for some time as to why in vitro systems reconstituted with homogeneous preparations of Pol II, general transcription factors (GTFs), and positive cofactor 4 (PC4) display relatively efficient activator-dependent (and basal) transcriptional activity (13,17). Although Mediator can further stimulate this activity, the basis of the high Mediatorindependent activity has remained unclear.…”

Cyclin-Dependent Kinase 8 Positively Cooperates with Mediator To Promote Thyroid Hormone Receptor-Dependent Transcriptional Activation

“…For instance, studies in yeast have revealed that the CDK8 module is associated with Mediator complexes in the genome upstream of active genes (2) and is essential for efficient transcriptional activation by both the Gal4 and Sip4 activators (19,24,57). Similarly, human Mediator complexes containing the CDK8 module are clearly recruited to target gene promoters in an activator-dependent manner both in vitro and in vivo and appear to promote, and in some cases are essential for, transcriptional activation (6,9,10,16,20,28,39,(58)(59)(60). Interestingly, CDK8 can specifically phosphorylate the CTD of Pol II (18,26,41,42,48), and CDK8 kinase activity in yeast specifically promotes Pol II transcription as well as the formation of a transcription reinitiation scaffold complex (26).…”