Malleable machines in transcription regulation: the mediator complex

Abstract

The Mediator complex provides an interface between gene-specific regulatory proteins and the general transcription machinery including RNA polymerase II (RNAP II). The complex has a modular architecture (Head, Middle, and Tail) and cryoelectron microscopy analysis suggested that it undergoes dramatic conformational changes upon interactions with activators and RNAP II. These rearrangements have been proposed to play a role in the assembly of the preinitiation complex and also to contribute to the regulatory mechanism of Mediator. In analogy to many regulatory and transcriptional proteins, we reasoned that Mediator might also utilize intrinsically disordered regions (IDRs) to facilitate structural transitions and transmit transcriptional signals. Indeed, a high prevalence of IDRs was found in various subunits of Mediator from both Saccharomyces cerevisiae and Homo sapiens, especially in the Tail and the Middle modules. The level of disorder increases from yeast to man, although in both organisms it significantly exceeds that of multiprotein complexes of a similar size. IDRs can contribute to Mediator's function in three different ways: they can individually serve as target sites for multiple partners having distinctive structures; they can act as malleable linkers connecting globular domains that impart modular functionality on the complex; and they can also facilitate assembly and disassembly of complexes in response to regulatory signals. Short segments of IDRs, termed molecular recognition features (MoRFs) distinguished by a high protein-protein interaction propensity, were identified in 16 and 19 subunits of the yeast and human Mediator, respectively. In Saccharomyces cerevisiae, the functional roles of 11 MoRFs have been experimentally verified, and those in the Med8/Med18/Med20 and Med7/Med21 complexes were structurally confirmed. Although the Saccharomyces cerevisiae and Homo sapiens Mediator sequences are only weakly conserved, the arrangements of the disordered regions and their embedded interaction sites are quite similar in the two organisms. All of these data suggest an integral role for intrinsic disorder in Mediator's function.

Figure 1. Mediator transmits regulatory signals from gene-specific activator proteins to the general transcription machinery, including RNA polymerase II (RNAP II, yellow), and general transcription factors (IIB, IID, IIE, IIF, IIH, light green).

The Tail interacts with a variety of activators/repressors and the regulatory signals are transferred via the Middle module to the Head that physically contacts RNAP II. The Middle also receives signals from the CDK module that dissociates prior to transcription. The shades of the blue colors correlate to the level of disorder in the different modules in Saccharomyces cerevisiae as computed in the present work.

Figure 2. Average disorder of the available Mediator subunits in Saccharomyces cerevisiae (grey) and in Homo sapiens (crosshatched) as computed by the IUPred algorithm .

0.5 (dashed line) is the threshold for disordered state and 0.4 (dotted line) is the average disorder of all disordered segments in the DisProt database . Subunits belonging to the different modules (Head, Middle, Tail, Cdk) are separated by vertical lines.

Figure 3. Amino acid compositions, relative to the set of globular proteins, of the Mediator (black), and its modules, Head (orange), Middle (green) and Tail (yellow) CDK (blue) in Saccharomyces cerevisiae (A) and in Homo sapiens (B).

Compositional profiling of intrinsically disordered proteins from the DisProt database is shown for comparison (red). The arrangement of the amino acids is by peak height for the set of disordered proteins from DisProt . Confidence intervals were estimated using per-protein bootstrapping with 1,000 iterations.

Figure 4. Abundance of IDRs in the Mediator complex and its modules.

The number of disordered segments of given length in Saccharomyces cerevisiae (grey) and in Homo sapiens (crosshatched) as computed by the IUPred algorithm is shown in the Mediator complex (A), in the Head (B), Middle (C) and Tail (D) modules.

Figure 5. Schematic representation of the Mediator complex: Head (orange), Middle (green), Tail (yellow), CDK (blue).

Subunits with higher than 50% average overall disorder (Med2, Med3 in Tail; Med9, Med19, Med26 in Middle and Med8 in Head) or subunits containing intrinsically disordered regions longer than 100 residues (Med12, Med13 of the CDK, Med1, Med9, Med26 of the Middle and Med15 of the Tail) in either Saccharomyces cerevisiae or in Homo sapiens are displayed by darker colors. Med19 and Med26 was assigned to the Middle module according to reference .

Figure 6. Location of α-MoRFs predicted by the PONDR VL-XT algorithm in Med7 and Med8 subunits of Saccharomyces cerevisiae in (A) Med7/Med21 (1yke) and (B) Med8/Med18/Med20 (2hzs) complexes.

The recognition motifs in Med7 (195–212) and Med8 (193–210) that are biased for an α-helical conformation in the bound state are shown by red.

Figure 7. Amino acid conservation of ordered (crosshatched) and disordered (gray) regions in Saccharomyces cerevisiae and in Homo sapiens.

Total amino acid conservation shown in black.

Figure 8. Conservation of disordered regions in Saccharomyces cerevisiae and in Homo sapiens.

The arrangement of ordered/disordered segments is compared to each other using positional (A) segmental overlap (B) measures on the actual Mediator protein sequences in MED_ALSEQ dataset (grey) and on the corresponding randomized MED_ALRAN dataset (crosshatched).