+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 3j0k | ||||||
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タイトル | Orientation of RNA polymerase II within the human VP16-Mediator-pol II-TFIIF assembly | ||||||
要素 |
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キーワード | TRANSFERASE/TRANSCRIPTION / TRANSFERASE-TRANSCRIPTION complex | ||||||
機能・相同性 | 機能・相同性情報 RPB4-RPB7 complex / RNA Polymerase I Transcription Initiation / Processing of Capped Intron-Containing Pre-mRNA / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / mRNA Capping / RNA polymerase II transcribes snRNA genes / TP53 Regulates Transcription of DNA Repair Genes ...RPB4-RPB7 complex / RNA Polymerase I Transcription Initiation / Processing of Capped Intron-Containing Pre-mRNA / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / mRNA Capping / RNA polymerase II transcribes snRNA genes / TP53 Regulates Transcription of DNA Repair Genes / termination of RNA polymerase II transcription / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Initiation And Promoter Clearance / RNA-templated transcription / RNA Polymerase II Pre-transcription Events / termination of RNA polymerase III transcription / Formation of TC-NER Pre-Incision Complex / transcription initiation at RNA polymerase III promoter / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / termination of RNA polymerase I transcription / RNA Polymerase I Promoter Escape / nucleolar large rRNA transcription by RNA polymerase I / Gap-filling DNA repair synthesis and ligation in TC-NER / transcription by RNA polymerase I / transcription initiation at RNA polymerase I promoter / Estrogen-dependent gene expression / nuclear-transcribed mRNA catabolic process / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / transcription by RNA polymerase III / RNA polymerase II activity / Dual incision in TC-NER / transcription elongation by RNA polymerase I / transcription-coupled nucleotide-excision repair / tRNA transcription by RNA polymerase III / RNA polymerase I activity / RNA polymerase I complex / RNA polymerase III complex / positive regulation of translational initiation / translesion synthesis / RNA polymerase II, core complex / translation initiation factor binding / DNA-templated transcription initiation / transcription initiation at RNA polymerase II promoter / transcription elongation by RNA polymerase II / P-body / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / cytoplasmic stress granule / mRNA processing / ribosome biogenesis / single-stranded DNA binding / transcription by RNA polymerase II / nucleic acid binding / single-stranded RNA binding / protein dimerization activity / mRNA binding / nucleotide binding / nucleolus / mitochondrion / DNA binding / zinc ion binding / nucleoplasm / nucleus / metal ion binding / cytoplasm 類似検索 - 分子機能 | ||||||
生物種 | Homo sapiens (ヒト) | ||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 36 Å | ||||||
データ登録者 | Bernecky, C. / Grob, P. / Ebmeier, C.C. / Nogales, E. / Taatjes, D.J. | ||||||
引用 | ジャーナル: PLoS Biol / 年: 2011 タイトル: Molecular architecture of the human Mediator-RNA polymerase II-TFIIF assembly. 著者: Carrie Bernecky / Patricia Grob / Christopher C Ebmeier / Eva Nogales / Dylan J Taatjes / 要旨: The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC), consists of multiple protein complexes and is approximately 3.5 MDa ...The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC), consists of multiple protein complexes and is approximately 3.5 MDa in size. At the heart of this assembly is the Mediator complex, which helps regulate PIC activity and interacts with the RNA polymerase II (pol II) enzyme. The structure of the human Mediator-pol II interface is not well-characterized, whereas attempts to structurally define the Mediator-pol II interaction in yeast have relied on incomplete assemblies of Mediator and/or pol II and have yielded inconsistent interpretations. We have assembled the complete, 1.9 MDa human Mediator-pol II-TFIIF complex from purified components and have characterized its structural organization using cryo-electron microscopy and single-particle reconstruction techniques. The orientation of pol II within this assembly was determined by crystal structure docking and further validated with projection matching experiments, allowing the structural organization of the entire human PIC to be envisioned. Significantly, pol II orientation within the Mediator-pol II-TFIIF assembly can be reconciled with past studies that determined the location of other PIC components relative to pol II itself. Pol II surfaces required for interacting with TFIIB, TFIIE, and promoter DNA (i.e., the pol II cleft) are exposed within the Mediator-pol II-TFIIF structure; RNA exit is unhindered along the RPB4/7 subunits; upstream and downstream DNA is accessible for binding additional factors; and no major structural re-organization is necessary to accommodate the large, multi-subunit TFIIH or TFIID complexes. The data also reveal how pol II binding excludes Mediator-CDK8 subcomplex interactions and provide a structural basis for Mediator-dependent control of PIC assembly and function. Finally, parallel structural analysis of Mediator-pol II complexes lacking TFIIF reveal that TFIIF plays a key role in stabilizing pol II orientation within the assembly. | ||||||
履歴 |
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-構造の表示
ムービー |
ムービービューア |
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構造ビューア | 分子: MolmilJmol/JSmol |
-ダウンロードとリンク
-ダウンロード
PDBx/mmCIF形式 | 3j0k.cif.gz | 789.7 KB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb3j0k.ent.gz | 641.6 KB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 3j0k.json.gz | ツリー表示 | PDBx/mmJSON形式 | |
その他 | その他のダウンロード |
-検証レポート
文書・要旨 | 3j0k_validation.pdf.gz | 842 KB | 表示 | wwPDB検証レポート |
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文書・詳細版 | 3j0k_full_validation.pdf.gz | 1.2 MB | 表示 | |
XML形式データ | 3j0k_validation.xml.gz | 142.5 KB | 表示 | |
CIF形式データ | 3j0k_validation.cif.gz | 212.4 KB | 表示 | |
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/j0/3j0k ftp://data.pdbj.org/pub/pdb/validation_reports/j0/3j0k | HTTPS FTP |
-関連構造データ
-リンク
-集合体
登録構造単位 |
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1 |
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-要素
-DNA-directed RNA polymerase II ... , 7種, 7分子 ABCDGIK
#1: タンパク質 | 分子量: 163180.016 Da / 分子数: 1 / 由来タイプ: 天然 / 由来: (天然) Homo sapiens (ヒト) / 細胞株: HeLa / Organelle: nucleus / 参照: UniProt: P04050*PLUS, DNA-directed RNA polymerase |
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#2: タンパク質 | 分子量: 138937.297 Da / 分子数: 1 / 由来タイプ: 天然 / 由来: (天然) Homo sapiens (ヒト) / 細胞株: HeLa / Organelle: nucleus / 参照: UniProt: P08518*PLUS, DNA-directed RNA polymerase |
#3: タンパク質 | 分子量: 30140.059 Da / 分子数: 1 / 由来タイプ: 天然 / 由来: (天然) Homo sapiens (ヒト) / 細胞株: HeLa / Organelle: nucleus / 参照: UniProt: P16370*PLUS, DNA-directed RNA polymerase |
#4: タンパク質 | 分子量: 25451.191 Da / 分子数: 1 / 由来タイプ: 天然 / 由来: (天然) Homo sapiens (ヒト) / 細胞株: HeLa / Organelle: nucleus / 参照: UniProt: P20433*PLUS, DNA-directed RNA polymerase |
#7: タンパク質 | 分子量: 19081.053 Da / 分子数: 1 / 由来タイプ: 天然 / 由来: (天然) Homo sapiens (ヒト) / 細胞株: HeLa / Organelle: nucleus / 参照: UniProt: P34087*PLUS, DNA-directed RNA polymerase |
#9: タンパク質 | 分子量: 14308.161 Da / 分子数: 1 / 由来タイプ: 天然 / 由来: (天然) Homo sapiens (ヒト) / 細胞株: HeLa / Organelle: nucleus / 参照: UniProt: P27999*PLUS, DNA-directed RNA polymerase |
#11: タンパク質 | 分子量: 13633.493 Da / 分子数: 1 / 由来タイプ: 天然 / 由来: (天然) Homo sapiens (ヒト) / 細胞株: HeLa / Organelle: nucleus / 参照: UniProt: P38902*PLUS, DNA-directed RNA polymerase |
-DNA-directed RNA polymerases I, II, and III ... , 4種, 4分子 EFHL
#5: タンパク質 | 分子量: 25117.094 Da / 分子数: 1 / 由来タイプ: 天然 / 由来: (天然) Homo sapiens (ヒト) / 細胞株: HeLa / Organelle: nucleus / 参照: UniProt: P20434*PLUS, DNA-directed RNA polymerase |
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#6: タンパク質 | 分子量: 9675.230 Da / 分子数: 1 / 由来タイプ: 天然 / 由来: (天然) Homo sapiens (ヒト) / 細胞株: HeLa / Organelle: nucleus / 参照: UniProt: P20435*PLUS, DNA-directed RNA polymerase |
#8: タンパク質 | 分子量: 16525.363 Da / 分子数: 1 / 由来タイプ: 天然 / 由来: (天然) Homo sapiens (ヒト) / 細胞株: HeLa / Organelle: nucleus / 参照: UniProt: P20436*PLUS, DNA-directed RNA polymerase |
#12: タンパク質・ペプチド | 分子量: 5252.261 Da / 分子数: 1 / 由来タイプ: 天然 / 由来: (天然) Homo sapiens (ヒト) / 細胞株: HeLa / Organelle: nucleus / 参照: DNA-directed RNA polymerase |
-タンパク質 , 1種, 1分子 J
#10: タンパク質 | 分子量: 8290.732 Da / 分子数: 1 / 由来タイプ: 天然 / 由来: (天然) Homo sapiens (ヒト) / 細胞株: HeLa / Organelle: nucleus / 参照: UniProt: P22139*PLUS, DNA-directed RNA polymerase |
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-非ポリマー , 2種, 10分子
#13: 化合物 | ChemComp-MG / |
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#14: 化合物 | ChemComp-ZN / |
-詳細
配列の詳細 | THIS ENTRY WAS MODELED WITH HOMOLOGOUS |
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-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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EM実験 | 試料の集合状態: PARTICLE / 3次元再構成法: 単粒子再構成法 |
-試料調製
構成要素 |
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分子量 | 値: 1.9 MDa / 実験値: NO | ||||||||||||||||||||||||||||||
緩衝液 | pH: 7.9 詳細: 20 mM HEPES, 0.10 mM EDTA, 150 mM KCl, 0.02% NP-40, 35% glycerol | ||||||||||||||||||||||||||||||
試料 | 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: 20 mM HEPES, 0.10 mM EDTA, 150 mM KCl, 0.02% NP-40, 35% glycerol | ||||||||||||||||||||||||||||||
染色 | タイプ: NEGATIVE 詳細: grids with adsorbed protein washed 3x with buffer containing 5% trehalose, 20 mM HEPES, 100 mM KCl, and 0.10 mM EDTA, then subjected to cryo-negative staining in a saturated solution (1.2M) ...詳細: grids with adsorbed protein washed 3x with buffer containing 5% trehalose, 20 mM HEPES, 100 mM KCl, and 0.10 mM EDTA, then subjected to cryo-negative staining in a saturated solution (1.2M) of ammonium molybdate (pH 7.5) 染色剤: ammonium molybdate | ||||||||||||||||||||||||||||||
試料支持 | 詳細: thin carbon-coated holey carbon 400 mesh copper grid | ||||||||||||||||||||||||||||||
急速凍結 | 凍結剤: ETHANE / Temp: 90 K 手法: blot for 2 seconds, dry for 3 seconds before plunging |
-電子顕微鏡撮影
実験機器 | モデル: Tecnai F20 / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TECNAI F20 |
電子銃 | 電子線源: FIELD EMISSION GUN / 加速電圧: 200 kV / 照射モード: FLOOD BEAM |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 29000 X / 最大 デフォーカス(公称値): 4500 nm / 最小 デフォーカス(公称値): 1000 nm / Cs: 2 mm / カメラ長: 0 mm |
試料ホルダ | 試料ホルダーモデル: GATAN LIQUID NITROGEN / 資料ホルダタイプ: side entry / 傾斜角・最大: 0 ° / 傾斜角・最小: 0 ° |
撮影 | 電子線照射量: 15 e/Å2 / フィルム・検出器のモデル: KODAK SO-163 FILM |
画像スキャン | サンプリングサイズ: 12.9 µm / デジタル画像の数: 106 / Od range: 1 / Quant bit size: 16 / Scanner model: OTHER |
放射 | プロトコル: SINGLE WAVELENGTH / 単色(M)・ラウエ(L): M / 散乱光タイプ: x-ray |
放射波長 | 相対比: 1 |
-解析
EMソフトウェア |
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CTF補正 | 詳細: each micrograph | ||||||||||||
対称性 | 点対称性: C1 (非対称) | ||||||||||||
3次元再構成 | 手法: multi-reference projection matching / 解像度: 36 Å / 解像度の算出法: FSC 0.5 CUT-OFF / 粒子像の数: 3146 / 詳細: The particles were selected interactively. / 対称性のタイプ: POINT | ||||||||||||
原子モデル構築 | プロトコル: RIGID BODY FIT / 空間: REAL / Target criteria: contour-based Laplacian correlation 詳細: REFINEMENT PROTOCOL--rigid body DETAILS--the TFIIS chain S was removed before fitting | ||||||||||||
原子モデル構築 | PDB-ID: 1Y1V | ||||||||||||
精密化ステップ | サイクル: LAST
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