An Activator Binding Module of Yeast RNA Polymerase II Holoenzyme

Abstract

The Mediator complex of Saccharomyces cerevisiae is required for both general and regulated transcription of RNA polymerase II (PolII) and is composed of two stable subcomplexes (Srb4 and Rgr1 subcomplexes). To decipher the function of each Mediator subcomplex and to delineate the functional relationship between the subcomplexes, we characterized the compositions and biochemical activities of PolII-Mediator complexes (holoenzymes) prepared from several Mediator mutant strains of S. cerevisiae. We found that holoenzymes devoid of a functional Gal11 module were defective for activated but not basal transcription in a reconstituted in vitro system. This activation-specific defect was correlated with a crippled physical interaction to transcriptional activator proteins, which could be bypassed by artificial recruitment of a mutant holoenzyme to a promoter. Consistent with this observation, a direct interaction between Gal11 and gene-specific transcriptional activator proteins was detected by far-Western analyses and column binding assays. In contrast, the srb5 deletion mutant holoenzyme was defective for both basal and activated transcription, despite its capacity for activator binding that is comparable to that of the wild-type holoenzyme. These results demonstrate that the Gal11 module of the Rgr1 subcomplex is required for the efficient recruitment of PolII holoenzyme to a promoter via activator-specific interactions, while the Srb4 subcomplex functions in the modulation of general polymerase activity.

Genetic Interactions Between Mediator and the Late G1-Specific Transcription Factor Swi6 in Saccharomyces cerevisiae

“…Mutations in the tail module components sin4 and pgd1 showed both growth defects and suppression when combined with swi6, but a third tail component, gal11, showed neither. It has been suggested that GAL11 mutants prevent association of other tail module subunits (Lee et al 1999;Park et al 2000). If that were the case, we would expect the gal11 phenotypes to include all the phenotypes associated with loss of Sin4 and Pgd1.…”

Drosophila homologs of transcriptional mediator complex subunits are required for adult cell and segment identity specification

“…It is not yet clear whether truly species-specific MED subunits exist. Still, the emergence or exchange of additional metazoan-specific subunits, as well as mixing and matching among various modular subcomplexes such as those detected in yeast (Lee and Kim 1998;Lee et al 1999), may have been an important element contributing to the diversification of genetic programs in complex organisms.…”

Two Distinct Roles of ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1) at Promoters and within Transcribed Regions of ATX1-Regulated Genes

“…Specifically, the formation of the preinitiation complex (PIC), transcription initiation, and the elongation and polyadenylation/termination stage each are associated with a distinct pattern of CTD phosphorylation. During the formation of PIC on the promoter DNA/chromatin template, transcription activators bind the coactivator protein complex, Mediator (Lee et al, 1999), which binds to the predominantly nonphosphorylated or hypophosphorylated form of Pol II and class II basal transcription factors (TFII). The TFIID component of PIC contains the TATA binding protein (TBP) that binds the TATA box sequence of promoters, as well as other TBP-associated factors (TAFs) or TBP-related factors involved in binding to promoters, particularly in those lacking a TATA box (Mü ller et al, 2007;Juven-Gershon and Kadonaga, 2010).…”