ジャーナル: Nucleic Acids Res / 年: 2014 タイトル: Functional domains of the 50S subunit mature late in the assembly process. 著者: Ahmad Jomaa / Nikhil Jain / Joseph H Davis / James R Williamson / Robert A Britton / Joaquin Ortega / 要旨: Despite the identification of many factors that facilitate ribosome assembly, the molecular mechanisms by which they drive ribosome biogenesis are poorly understood. Here, we analyze the late stages ...Despite the identification of many factors that facilitate ribosome assembly, the molecular mechanisms by which they drive ribosome biogenesis are poorly understood. Here, we analyze the late stages of assembly of the 50S subunit using Bacillus subtilis cells depleted of RbgA, a highly conserved GTPase. We found that RbgA-depleted cells accumulate late assembly intermediates bearing sub-stoichiometric quantities of ribosomal proteins L16, L27, L28, L33a, L35 and L36. Using a novel pulse labeling/quantitative mass spectrometry technique, we show that this particle is physiologically relevant and is capable of maturing into a complete 50S particle. Cryo-electron microscopy and chemical probing revealed that the central protuberance, the GTPase associating region and tRNA-binding sites in this intermediate are unstructured. These findings demonstrate that key functional sites of the 50S subunit remain unstructured until late stages of maturation, preventing the incomplete subunit from prematurely engaging in translation. Finally, structural and biochemical analysis of a ribosome particle depleted of L16 indicate that L16 binding is necessary for the stimulation of RbgA GTPase activity and, in turn, release of this co-factor, and for conversion of the intermediate to a complete 50S subunit.
pH: 7.5 詳細: 10 mM Tris-HCl, pH 7.5, 10 mM magnesium acetate, 60 mM ammonium chloride, 3 mM 2-mercaptoethanol
グリッド
詳細: 400 mesh holey carbon grids with an additional layer (5-10 nm) of thin carbon. Grids were glow discharged at 5 mA for 15 seconds.
凍結
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 77 K / 装置: FEI VITROBOT MARK III 手法: Grids were blotted twice, for 7 seconds each time, before being plunged into liquid ethane.
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電子顕微鏡法
顕微鏡
JEOL 2010F
温度
最低: 77 K / 最高: 77 K
アライメント法
Legacy - 非点収差: Objective lens astigmatism was corrected at 300,000 times magnification
日付
2012年2月1日
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: NIKON SUPER COOLSCAN 9000 デジタル化 - サンプリング間隔: 12.7 µm / 実像数: 300 / 平均電子線量: 20 e/Å2 / ビット/ピクセル: 8