ジャーナル: Structure / 年: 2010 タイトル: Structural underpinnings of nitrogen regulation by the prototypical nitrogen-responsive transcriptional factor NrpR. 著者: Goragot Wisedchaisri / David M Dranow / Thomas J Lie / Jeffrey B Bonanno / Yury Patskovsky / Sinem A Ozyurt / J Michael Sauder / Steven C Almo / Stephen R Wasserman / Stephen K Burley / John ...著者: Goragot Wisedchaisri / David M Dranow / Thomas J Lie / Jeffrey B Bonanno / Yury Patskovsky / Sinem A Ozyurt / J Michael Sauder / Steven C Almo / Stephen R Wasserman / Stephen K Burley / John A Leigh / Tamir Gonen / 要旨: Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. ...Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. Cellular 2OG concentrations increase during nitrogen starvation. We recently identified a family of 2OG-sensing proteins--the nitrogen regulatory protein NrpR--that bind DNA and repress transcription of nitrogen assimilation genes. We used X-ray crystallography to determine the structure of NrpR regulatory domain. We identified the NrpR 2OG-binding cleft and show that residues predicted to interact directly with 2OG are conserved among diverse classes of 2OG-binding proteins. We show that high levels of 2OG inhibit NrpRs ability to bind DNA. Electron microscopy analyses document that NrpR adopts different quaternary structures in its inhibited 2OG-bound state compared with its active apo state. Our results indicate that upon 2OG release, NrpR repositions its DNA-binding domains correctly for optimal interaction with DNA thereby enabling gene repression.
pH: 7.5 詳細: 100mM Tris HCl pH 7.5, 1M KCl and 5mM glycerol, 10mM 2-oxoglutarate
染色
タイプ: NEGATIVE 詳細: A 2 microlitre drop of NrpR was applied to a carbon-coated grid, washed 3 times with milliQ water and stained using 0.075% uranyl formate.
凍結
凍結剤: NONE / 装置: OTHER
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電子顕微鏡法
顕微鏡
FEI TECNAI 12
撮影
デジタル化 - スキャナー: NIKON SUPER COOLSCAN 9000
電子線
加速電圧: 120 kV / 電子線源: LAB6
電子光学系
照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / 倍率(公称値): 52000
試料ステージ
試料ホルダー: Eucentric / 試料ホルダーモデル: OTHER
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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: 22.0 Å / 解像度の算出法: FSC 0.5 CUT-OFF / ソフトウェア - 名称: Spider / 詳細: Random conical tilt followed by angular refinement / 使用した粒子像数: 8498