Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Apr 13;417(5):387-94.
doi: 10.1016/j.jmb.2012.02.014. Epub 2012 Feb 16.

Activator-mediator binding stabilizes RNA polymerase II orientation within the human mediator-RNA polymerase II-TFIIF assembly

Carrie Bernecky  1 , Dylan J Taatjes
Affiliations

Activator-mediator binding stabilizes RNA polymerase II orientation within the human mediator-RNA polymerase II-TFIIF assembly

Carrie Bernecky et al. J Mol Biol. .

Abstract

The human Mediator complex controls RNA polymerase II (pol II) function in ways that remain incompletely understood. Activator-Mediator binding alters Mediator structure, and these activator-induced structural shifts appear to play key roles in regulating transcription. A recent cryo-electron microscopy (EM) analysis revealed that pol II adopted a stable orientation within a Mediator-pol II-TFIIF assembly in which Mediator was bound to the activation domain of viral protein 16 (VP16). Whereas TFIIF was shown to be important for orienting pol II within this assembly, the potential role of the activator was not assessed. To determine how activator binding might affect pol II orientation, we isolated human Mediator-pol II-TFIIF complexes in which Mediator was not bound to an activator. Cryo-EM analysis of this assembly, coupled with pol II crystal structure docking, revealed that pol II binds Mediator at the same general location; however, in contrast to VP16-bound Mediator, pol II does not appear to stably orient in the absence of an activator. Variability in pol II orientation might be important mechanistically, perhaps to enable sense and antisense transcription at human promoters. Because Mediator interacts extensively with pol II, these results suggest that Mediator structural shifts induced by activator binding help stably orient pol II prior to transcription initiation.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Purification of (A) pol II, (B) Mediator, and (C) TFIIF. In each case, a schematic of the purification protocol is shown at left, with gels showing the purified complex at the right. Pol II and Mediator gels were stained with silver whereas TFIIF was stained with coomassie. Identities of subunits are shown at the right of each gel.
Fig. 2
Fig. 2
Isolation of the Mediator–pol II–TFIIF assembly. (A) Schematic of the protocol. The gradient was set up such that the complete Mediator–pol II–TFIIF assembly would migrate and concentrate in the final fraction. Note that Mediator is not bound to an activator in these experiments. (B) Silver-stained gel of the glycerol gradient fractions. Free TFIIF, free Mediator, and free pol II migrate earlier in the gradient, as indicated. Subunit identities shown at the right. (C) Western blot analysis confirms the presence of Mediator, pol II, and TFIIF in the final gradient fraction, as expected. The pol II antibody was directed against RPB1, the Mediator antibody targeted MED23, and the TFIIF antibody RAP74.
Fig. 3
Fig. 3
Three-dimensional structure and pol II docking results for the activator-free Mediator–pol II–TFIIF assembly. (A) Different views of the cryo-EM reconstruction, rendered to 1.8 MDa. Rotation of the structure shown at left. The resolution of this structure is 32 Å, based upon both the Fourier Shell Correlation (0.5 cutoff) and the half-bit threshold., Asterisks for the “front” view represent sites where the pol II stalk protrudes from the structure, based upon 1) extra protein density observed at higher mass thresholds (not shown), and 2) the top pol II docking results based upon cross-correlation coefficients with 8 different pol II crystal structures (see Supplemental Table 1). The absence of clear pol II stalk density at 1.8MDa rendering is consistent with multiple pol II orientations within the sample (see text). The cryo-EM map has been deposited in the EM Databank (http://www.ebi.ac.uk/pdbe/emdb/), entry number 5344.(B) General location of pol II, based upon the docking experiments. (C) The 3D variance map for Mediator–pol II–TFIIF. The variance map is displayed in blue and superimposed upon the Mediator–pol II–TFIIF cryo-EM map, which is shown in green mesh. To enhance visualization, peaks in the 3D variance map are also marked with purple spheres with a radius of 5 pixels (21 Å).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Taatjes DJ, Naar AM, Andel F, Nogales E, Tjian R. Structure, function, and activator-induced conformations of the CRSP coactivator. Science. 2002;295:1058–1062. - PubMed
    1. Taatjes DJ, Schneider-Poetsch T, Tjian R. Distinct conformational states of nuclear receptor-bound CRSP-Med complexes. Nat StructMolBiol. 2004;11:664–671. - PubMed
    1. Meyer KD, Lin S, Bernecky C, Gao Y, Taatjes DJ. p53 activates transcription by directing structural shifts in Mediator. Nat StructMolBiol. 2010;17:753–760. - PMC - PubMed
    1. Fondell JD, Ge H, Roeder RG. Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex. Proc. Natl. Acad. Sci. USA. 1996;93:8329–8333. - PMC - PubMed
    1. Naar AM, Beaurang PA, Zhou S, Abraham S, Solomon W, Tjian R. Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature. 1999;398:828–832. - PubMed

Publication types

MeSH terms

LinkOut - more resources

-