ジャーナル: Nature / 年: 2010 タイトル: TFIIA and the transactivator Rap1 cooperate to commit TFIID for transcription initiation. 著者: Gabor Papai / Manish K Tripathi / Christine Ruhlmann / Justin H Layer / P Anthony Weil / Patrick Schultz / 要旨: Transcription of eukaryotic messenger RNA (mRNA) encoding genes by RNA polymerase II (Pol II) is triggered by the binding of transactivating proteins to enhancer DNA, which stimulates the recruitment ...Transcription of eukaryotic messenger RNA (mRNA) encoding genes by RNA polymerase II (Pol II) is triggered by the binding of transactivating proteins to enhancer DNA, which stimulates the recruitment of general transcription factors (TFIIA, B, D, E, F, H) and Pol II on the cis-linked promoter, leading to pre-initiation complex formation and transcription. In TFIID-dependent activation pathways, this general transcription factor containing TATA-box-binding protein is first recruited on the promoter through interaction with activators and cooperates with TFIIA to form a committed pre-initiation complex. However, neither the mechanisms by which activation signals are communicated between these factors nor the structural organization of the activated pre-initiation complex are known. Here we used cryo-electron microscopy to determine the architecture of nucleoprotein complexes composed of TFIID, TFIIA, the transcriptional activator Rap1 and yeast enhancer-promoter DNA. These structures revealed the mode of binding of Rap1 and TFIIA to TFIID, as well as a reorganization of TFIIA induced by its interaction with Rap1. We propose that this change in position increases the exposure of TATA-box-binding protein within TFIID, consequently enhancing its ability to interact with the promoter. A large Rap1-dependent DNA loop forms between the activator-binding site and the proximal promoter region. This loop is topologically locked by a TFIIA-Rap1 protein bridge that folds over the DNA. These results highlight the role of TFIIA in transcriptional activation, define a molecular mechanism for enhancer-promoter communication and provide structural insights into the pathways of intramolecular communication that convey transcription activation signals through the TFIID complex.
pH: 7.9 / 詳細: 10 mM Tris-HCl pH 7.9, 300 mM KOAc and 5 mM MgCl2
グリッド
詳細: 300 mesh Cu/Rh holey grid
凍結
凍結剤: ETHANE / チャンバー内湿度: 95 % / チャンバー内温度: 80 K / 装置: OTHER / 詳細: Vitrification instrument: Vitrobot
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電子顕微鏡法
顕微鏡
FEI POLARA 300
温度
平均: 81 K
撮影
カテゴリ: FILM / フィルム・検出器のモデル: KODAK SO-163 FILM / デジタル化 - スキャナー: PRIMESCAN / デジタル化 - サンプリング間隔: 5.1 µm / 実像数: 90 / 平均電子線量: 16 e/Å2 / Od range: 1.4
電子線
加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN
電子光学系
倍率(補正後): 38950 / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2.0 mm / 倍率(公称値): 39000
試料ステージ
試料ホルダー: Eucentric / 試料ホルダーモデル: OTHER
実験機器
モデル: Tecnai Polara / 画像提供: FEI Company
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画像解析
CTF補正
詳細: Each particle
最終 再構成
アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 24.5 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: Imagic, Spider 詳細: The map was reconstructed from 3-D MSA separated dataset. 使用した粒子像数: 26901