ジャーナル: Nature / 年: 2006 タイトル: Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit. 著者: Thorsten Schäfer / Bohumil Maco / Elisabeth Petfalski / David Tollervey / Bettina Böttcher / Ueli Aebi / Ed Hurt / 要旨: The formation of eukaryotic ribosomes is a multistep process that takes place successively in the nucleolar, nucleoplasmic and cytoplasmic compartments. Along this pathway, multiple pre-ribosomal ...The formation of eukaryotic ribosomes is a multistep process that takes place successively in the nucleolar, nucleoplasmic and cytoplasmic compartments. Along this pathway, multiple pre-ribosomal particles are generated, which transiently associate with numerous non-ribosomal factors before mature 60S and 40S subunits are formed. However, most mechanistic details of ribosome biogenesis are still unknown. Here we identify a maturation step of the yeast pre-40S subunit that is regulated by the protein kinase Hrr25 and involves ribosomal protein Rps3. A high salt concentration releases Rps3 from isolated pre-40S particles but not from mature 40S subunits. Electron microscopy indicates that pre-40S particles lack a structural landmark present in mature 40S subunits, the 'beak'. The beak is formed by the protrusion of 18S ribosomal RNA helix 33, which is in close vicinity to Rps3. Two protein kinases Hrr25 and Rio2 are associated with pre-40S particles. Hrr25 phosphorylates Rps3 and the 40S synthesis factor Enp1. Phosphorylated Rsp3 and Enp1 readily dissociate from the pre-ribosome, whereas subsequent dephosphorylation induces formation of the beak structure and salt-resistant integration of Rps3 into the 40S subunit. In vivo depletion of Hrr25 inhibits growth and leads to the accumulation of immature 40S subunits that contain unstably bound Rps3. We conclude that the kinase activity of Hrr25 regulates the maturation of 40S ribosomal subunits.
凍結剤: ETHANE / チャンバー内湿度: 90 % / チャンバー内温度: 295 K / 装置: HOMEMADE PLUNGER 詳細: Vitrification instrument: Plunger with environmental chamber 手法: Blot for 15 s with Whatman No 1
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電子顕微鏡法
顕微鏡
FEI/PHILIPS CM200FEG
温度
平均: 94 K
アライメント法
Legacy - 非点収差: bjective lens astigmatism was corrected at
236 out of 400 classes were used for final reconstruction
CTF補正
詳細: Each Particle
最終 再構成
想定した対称性 - 点群: C1 (非対称) / アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 32.0 Å / 解像度の算出法: FSC 0.5 CUT-OFF / ソフトウェア - 名称: IMAGIC 5 詳細: reconstructions were calculated from half of the class averages. Euler angles of class averages were determined by projection matching 使用した粒子像数: 4827