Architecture of an RNA Polymerase II Transcription Pre-Initiation Complex

Murakami, Kenji; Elmlund, Hans; Kalisman, Nir; Bushnell, David; Adams, Christopher M.; Azubel, Maia; Elmlund, Dominika; Levi‐Kalisman, Yael; Liu, Xin; Gibbons, Brian; Levitt, Michael; Kornberg, Roger D.

doi:10.1126/science.1238724

Cited by 149 publications

(149 citation statements)

References 64 publications

(97 reference statements)

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“…Furthermore, the C‐terminal cyclin domain of Rrn7 is not positioned as closely to the DNA as in Pol II, where the C‐terminal cyclin domain of TFIIB contacts DNA upstream of the TATA box (Plaschka et al , 2016). The observed differences could be a result of DNA bending by TBP in the Pol II PIC, which brings the DNA in close proximity to the C‐terminal cyclin domain (Murakami et al , 2013, 2015; Plaschka et al , 2015, 2016; He et al , 2016). Nevertheless, the overall conformation of the C‐terminal cyclin domain relative to the N‐terminal cyclin domain is conserved between TFIIB‐like factors (Fig 3C).…”

Section: Resultsmentioning

confidence: 99%

“…Pol I contributes more than 60% to the total cellular transcription activity of the cell and accordingly requires high transcription initiation rates (Schneider, 2012). During transcription initiation, Pol I, similar to other eukaryotic RNA polymerases, assembles together with its specific transcription factors on the ribosomal RNA promoter to form the pre‐initiation complex (PIC; Murakami et al , 2013). In sequential steps, the PIC transitions from a closed complex (CC), in which the DNA is still double‐stranded, to an open complex (OC), in which the double‐stranded DNA in the vicinity of the transcription start site is melted.…”

Section: Introductionmentioning

confidence: 99%

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Structural insights into transcription initiation by yeast RNA polymerase I

et al. 2017

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In eukaryotic cells, RNA polymerase I (Pol I) synthesizes precursor ribosomal RNA (pre‐rRNA) that is subsequently processed into mature rRNA. To initiate transcription, Pol I requires the assembly of a multi‐subunit pre‐initiation complex (PIC) at the ribosomal RNA promoter. In yeast, the minimal PIC includes Pol I, the transcription factor Rrn3, and Core Factor (CF) composed of subunits Rrn6, Rrn7, and Rrn11. Here, we present the cryo‐EM structure of the 18‐subunit yeast Pol I PIC bound to a transcription scaffold. The cryo‐EM map reveals an unexpected arrangement of the DNA and CF subunits relative to Pol I. The upstream DNA is positioned differently than in any previous structures of the Pol II PIC. Furthermore, the TFIIB‐related subunit Rrn7 also occupies a different location compared to the Pol II PIC although it uses similar interfaces as TFIIB to contact DNA. Our results show that although general features of eukaryotic transcription initiation are conserved, Pol I and Pol II use them differently in their respective transcription initiation complexes.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Structural insights into transcription initiation by yeast RNA polymerase I

et al. 2017

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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

347

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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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“…Indeed, today we understand the regulation of Pol II initiation by the general transcription factors (GTFs) at a detailed molecular level. 1,2 During the past several years, biochemical and genomic analyses have revealed that Pol II recruitment is not the only point of transcriptional regulation. Rather, the transition between initiation and elongation is a multi-step process where any point between recruitment and productive elongation can potentially be regulated for proper gene expression in eukaryotes.…”

Section: Introductionmentioning

confidence: 99%