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Structure of a Complete Mediator-RNA Polymerase II Pre-Initiation Complex.

ABSTRACT: A complete, 52-protein, 2.5 million dalton, Mediator-RNA polymerase II pre-initiation complex (Med-PIC) was assembled and analyzed by cryo-electron microscopy and by chemical cross-linking and mass spectrometry. The resulting complete Med-PIC structure reveals two components of functional significance, absent from previous structures, a protein kinase complex and the Mediator-activator interaction region. It thereby shows how the kinase and its target, the C-terminal domain of the polymerase, control Med-PIC interaction and transcription.

SUBMITTER: Robinson PJ

PROVIDER: S-EPMC5589196 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Structure of a Complete Mediator-RNA Polymerase II Pre-Initiation Complex.

Robinson Philip J PJ Trnka Michael J MJ Bushnell David A DA Davis Ralph E RE Mattei Pierre-Jean PJ Burlingame Alma L AL Kornberg Roger D RD

Cell 20160901 6

A complete, 52-protein, 2.5 million dalton, Mediator-RNA polymerase II pre-initiation complex (Med-PIC) was assembled and analyzed by cryo-electron microscopy and by chemical cross-linking and mass spectrometry. The resulting complete Med-PIC structure reveals two components of functional significance, absent from previous structures, a protein kinase complex and the Mediator-activator interaction region. It thereby shows how the kinase and its target, the C-terminal domain of the polymerase, co ...[more]

PMID: 27610567

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Structure of a Complete Mediator-RNA Polymerase II Pre-Initiation Complex, and the Regulation of Transcription

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Architecture of the RNA polymerase II-Mediator core transcription initiation complex

Project description:Comparative Dynamic Transcriptome Analysis (cDTA) enables global analysis of newly synthesized RNA as described in Sun et al. Genome Res. 2012 (DOI:10.1101/gr.130161.111) and reveals defects in transcription with much higher sensitivity than conventional steady-state methods. cDTA was carried out as described in Sun et al. Genome Res. 2012 (DOI:10.1101/gr.130161.111), using the S. cerevisiae heterozygous Med17/med17delta strain (Euroscarf) transfected with plasmids pRS315-SRB4 or pRS315-srb4-ts as described in Larivi̬re et al. Nature. 2012 (DOI:10.1038/nature11670), and Y40343-wildtype (Euroscarf) or Med18-FRB-KanMX6 (Euroscarf) strains. Heatshock of SRB4 and srb4-ts strains was applied for 18 or 60 minutes at 37C prior to RNA labeling as described in Sun et al. Genome Res. 2012 (DOI:10.1101/gr.130161.111). To deplete the Med18 subunit from the nucleus, anchor-away experiments were performed by rapamycin treatment (1 ug/ml in 200 mL YPD) for 18 or 60 minutes at 30C prior to RNA labeling as described in Sun et al. Mol. Cell. 2013 (DOI:10.1016/j.molcel.2013.09.010). Data analysis was as described in Sun et al. Genome Res. 2012 (DOI:10.1101/gr.130161.111).

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Project description:Previous x-ray crystal structures have given insight into the mechanism of transcription and the role of general transcription factors in the initiation of the process. A structure of an RNA polymerase II-general transcription factor TFIIB complex at 4.5 angstrom resolution revealed the amino-terminal region of TFIIB, including a loop termed the "B finger," reaching into the active center of the polymerase where it may interact with both DNA and RNA, but this structure showed little of the carboxyl-terminal region. A new crystal structure of the same complex at 3.8 angstrom resolution obtained under different solution conditions is complementary with the previous one, revealing the carboxyl-terminal region of TFIIB, located above the polymerase active center cleft, but showing none of the B finger. In the new structure, the linker between the amino- and carboxyl-terminal regions can also be seen, snaking down from above the cleft toward the active center. The two structures, taken together with others previously obtained, dispel long-standing mysteries of the transcription initiation process.

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Structure of the TFIIIC subcomplex τA provides insights into RNA polymerase III pre-initiation complex formation.

Project description:Transcription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it is also involved in chromatin organization and regulation of Pol II genes through interaction with CTCF and condensin II. Here, we report the structure of the S. cerevisiae TFIIIC subcomplex τA, which contains the most conserved subunits of TFIIIC and is responsible for recruitment of TFIIIB and transcription start site (TSS) selection at Pol III genes. We show that τA binding to its promoter is auto-inhibited by a disordered acidic tail of subunit τ95. We further provide a negative-stain reconstruction of τA bound to the TFIIIB subunits Brf1 and TBP. This shows that a ruler element in τA achieves positioning of TFIIIB upstream of the TSS, and suggests remodeling of the complex during assembly of TFIIIB by TFIIIC.

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Yeast PIC-Mediator structure with RNA polymerase II C-terminal domain.

Project description:For transcription initiation, RNA polymerase II (Pol II) forms a preinitiation complex (PIC) that associates with the general coactivator Mediator. Whereas atomic models of the human PIC-Mediator structure have been reported, structures for its yeast counterpart remain incomplete. Here, we present an atomic model for the yeast PIC with core Mediator, including the Mediator middle module that was previously poorly resolved and including subunit Med1 that was previously lacking. We observe three peptide regions containing eleven of the 26 heptapeptide repeats of the flexible C-terminal repeat domain (CTD) of Pol II. Two of these CTD regions bind between the Mediator head and middle modules and form defined CTD-Mediator interactions. CTD peptide 1 binds between the Med6 shoulder and Med31 knob domains, whereas CTD peptide 2 forms additional contacts with Med4. The third CTD region (peptide 3) binds in the Mediator cradle and associates with the Mediator hook. Comparisons with the human PIC-Mediator structure show that the central region in peptide 1 is similar and forms conserved contacts with Mediator, whereas peptides 2 and 3 exhibit distinct structures and Mediator interactions.

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Project description:Notwithstanding numerous published structures of RNA Polymerase II (Pol II), structural details of Pol II engaging a complete nucleic acid scaffold have been lacking. Here, we report the structures of TFIIF-stabilized transcribing Pol II complexes, revealing the upstream duplex and full transcription bubble. The upstream duplex lies over a wedge-shaped loop from Rpb2 that engages its minor groove, providing part of the structural framework for DNA tracking during elongation. At the upstream transcription bubble fork, rudder and fork loop 1 residues spatially coordinate strand annealing and the nascent RNA transcript. At the downstream fork, a network of Pol II interactions with the non-template strand forms a rigid domain with the trigger loop (TL), allowing visualization of its open state. Overall, our observations suggest that "open/closed" conformational transitions of the TL may be linked to interactions with the non-template strand, possibly in a synchronized ratcheting manner conducive to polymerase translocation.

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