Department of Molecular Biology, Skaggs Institute for Chemical Biology, The Scripps Research Institute, MB-33, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
J Mol Biol. 2010 Oct 29;403(3):331-45. doi: 10.1016/j.jmb.2010.08.005. Epub 2010 Aug 13.
Although high-resolution structures of the ribosome have been solved in a series of functional states, relatively little is known about how the ribosome assembles, particularly in vivo. Here, a general method is presented for studying the dynamics of ribosome assembly and ribosomal assembly intermediates. Since significant quantities of assembly intermediates are not present under normal growth conditions, the antibiotic neomycin is used to perturb wild-type Escherichia coli. Treatment of E. coli with the antibiotic neomycin results in the accumulation of a continuum of assembly intermediates for both the 30S and 50S subunits. The protein composition and the protein stoichiometry of these intermediates were determined by quantitative mass spectrometry using purified unlabeled and (15)N-labeled wild-type ribosomes as external standards. The intermediates throughout the continuum are heterogeneous and are largely depleted of late-binding proteins. Pulse-labeling with (15)N-labeled medium time-stamps the ribosomal proteins based on their time of synthesis. The assembly intermediates contain both newly synthesized proteins and proteins that originated in previously synthesized intact subunits. This observation requires either a significant amount of ribosome degradation or the exchange or reuse of ribosomal proteins. These specific methods can be applied to any system where ribosomal assembly intermediates accumulate, including strains with deletions or mutations of assembly factors. This general approach can be applied to study the dynamics of assembly and turnover of other macromolecular complexes that can be isolated from cells.
尽管核糖体的高分辨率结构已在一系列功能状态下得到解决，但对于核糖体如何组装，特别是在体内组装，人们知之甚少。在这里，提出了一种研究核糖体组装动力学和核糖体组装中间产物的通用方法。由于在正常生长条件下，大量的组装中间产物不存在，因此使用抗生素新霉素来干扰野生型大肠杆菌。用抗生素新霉素处理大肠杆菌会导致 30S 和 50S 亚基的组装中间体连续积累。通过使用纯化的未标记和（15）N 标记的野生型核糖体作为外部标准，通过定量质谱法确定这些中间体的蛋白质组成和蛋白质化学计量。这些中间产物在整个连续体中都是异质的，并且很大程度上缺乏晚期结合蛋白。用（15）N 标记的培养基进行脉冲标记，根据蛋白质的合成时间对核糖体蛋白进行时间标记。组装中间体既包含新合成的蛋白质，也包含源自先前合成的完整亚基的蛋白质。这一观察结果需要大量的核糖体降解，或者需要核糖体蛋白的交换或再利用。这些特定的方法可以应用于任何组装中间产物积累的系统，包括组装因子缺失或突变的菌株。这种通用方法可用于研究可从细胞中分离出的其他大分子复合物的组装和周转率的动力学。
