Conserved architecture of the core RNA polymerase II initiation complex

Mühlbacher, Wolfgang; Sainsbury, Sarah; Hemann, M.; Hantsche, M.; Neyer, Simon; Herzog, Franz; Cramer, Patrick

doi:10.1038/ncomms5310

Cited by 39 publications

(39 citation statements)

References 36 publications

(73 reference statements)

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“…The computational models in Figure 6 also imply that p53 bound to Pol II would be in close proximity to the binding site of the core initiation machinery, specifically the GTFs TFIID and TFIIB, (Geiger et al 1996;Nikolov et al 1996;Kostrewa et al 2009;Muhlbacher et al 2014). This observation is supported by several lines of evidence.…”

Section: Discussionsupporting

confidence: 69%

Structural visualization of the p53/RNA polymerase II assembly

et al. 2016

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The master tumor suppressor p53 activates transcription in response to various cellular stresses in part by facilitating recruitment of the transcription machinery to DNA. Recent studies have documented a direct yet poorly characterized interaction between p53 and RNA polymerase II (Pol II). Therefore, we dissected the human p53/Pol II interaction via single-particle cryo-electron microscopy, structural docking, and biochemical analyses. This study reveals that p53 binds Pol II via the Rpb1 and Rpb2 subunits, bridging the DNA-binding cleft of Pol II proximal to the upstream DNA entry site. In addition, the key DNA-binding surface of p53, frequently disrupted in various cancers, remains exposed within the assembly. Furthermore, the p53/Pol II cocomplex displays a closed conformation as defined by the position of the Pol II clamp domain. Notably, the interaction of p53 and Pol II leads to increased Pol II elongation activity. These findings indicate that p53 may structurally regulate DNA-binding functions of Pol II via the clamp domain, thereby providing insights into p53-regulated Pol II transcription.

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Section: Discussionsupporting

confidence: 69%

Structural visualization of the p53/RNA polymerase II assembly

et al. 2016

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“…RNA synthesis leads to the initially transcribing complex (ITC) and later to release of the general factors and formation of the elongation complex. Recent studies elucidated the three-dimensional architecture of initiation intermediates, including the ITC [3][4][5][6][7][8][9] .…”

mentioning

confidence: 99%

“…cMed corresponds to the endogenous core Mediator purified from yeast 23 , and has a human counterpart that was reported while our manuscript was in revision 24 . cMed binds the core ITC (cITC), which lacks TFIIE and TFIIH and is stabilized by a short six-nucleotide RNA 6,9 . We resolved the architecture of the cITC-cMed complex by cryo-EM and protein crosslinking.…”

mentioning

confidence: 99%

Architecture of the RNA polymerase II–Mediator core initiation complex

Plaschka

Larivière

Wenzeck

et al. 2015

Nature

282

351

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The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.

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“…This includes information about the subunit organization of the polymerases themselves as well as the organization of polymerases with additional proteins into higher-order functional units. Studies have targeted RNA Pol I [81,82], II [83], and III [84] as well as the Pol II-PIC complex [85,86] and the Pol II-capping enzyme complex [87]. A subcomplex of the Mediator complex (the so-called middle module) [88] and the Mediator head module in complex with the C-terminal domain of Pol II [89] were also investigated by XL-MS. More recently, XL-MS was used to provide spatial restraints on the Mediator core and the Pol II-Mediator core initiation complex in the most comprehensive study on polymerases and associated complexes to date [90].…”

Section: Ribosomes and Associated Proteinsmentioning

confidence: 99%

Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

Leitner

Faini

Stengel

et al. 2016

Trends in Biochemical Sciences

340

285

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In recent years, chemical crosslinking of protein complexes and the identification of crosslinked residues by mass spectrometry (XL-MS; sometimes abbreviated as CX-MS) has become an important technique bridging mass spectrometry (MS) and structural biology. By now, XL-MS is well established and supported by publicly available resources as a convenient and versatile part of the structural biologist's toolbox. The combination of XL-MS with cryoelectron microscopy (cryo-EM) and/or integrative modeling is particularly promising to study the topology and structure of large protein assemblies. Among the targets studied so far are proteasomes, ribosomes, polymerases, chromatin remodelers, and photosystem complexes. Here we provide an overview of recent advances in XL-MS, the current state of the field, and a cursory outlook on future challenges. Chemical Crosslinking as a Tool for Structural BiologyStructural biology makes use of many different techniques to elucidate the 3D structures of proteins and protein complexes. While high-resolution structures have traditionally been obtained by X-ray crystallography, cryo-EM is increasingly able to also generate (near) atomic-resolution models. In recent years, techniques and applications of MS have also rapidly progressed. Earlier studies were largely focused on the large-scale identification and quantification of proteins, whereas recent methods also support queries into the composition, stoichiometry, and spatial arrangement of subunits in a complex. These developments have now further progressed toward generating information that contributes, as part of hybrid structural strategies, to the structure elucidation of large molecular assemblies including protein complexes that perform essential processes in the cell. XL-MS is a particularly powerful mass spectrometric technique in this respect, because it provides several layers of information. Identifying protein-protein contacts through XL-MS confirms physical proximity between subunits because the proteins must be close enough in space to be crosslinked. Localizing the side chains that are connected restricts this proximity to certain regions (e.g., domains or even single helices or loops). Finally, the structure of the connected side chains and the crosslinker moiety impart a distance restraint that can be used for molecular modeling purposes because an upper Trends Chemical crosslinking followed by the mass spectrometric analysis of cross linked peptides (XL MS) identifies con tact sites between residues within a single or between multiple proteins.The application of XL MS to many bio logically relevant molecular machines has been shown, with a rapidly growing number of successful studies reported in the past 2 3 years.Crosslinking data are useful in integra tive modeling workflows by providing distance restraints on the surface of folded proteins and complexes. XL MS has been shown to be particularly powerful in combination with 3D cryo electron microscopy. Sciences ; 41 (2016), 1. -S. 20-32 https://dx.doi.org/10.1016...

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Conserved architecture of the core RNA polymerase II initiation complex

Cited by 39 publications

References 36 publications

Structural visualization of the p53/RNA polymerase II assembly

Structural visualization of the p53/RNA polymerase II assembly

Architecture of the RNA polymerase II–Mediator core initiation complex

Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

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