在活跃生长的细胞中,翻译启动的速度相对较快。因此,同时参与翻译的多个核糖体沿着单个mRNA分子间隔开。这些结构,称为多体,可以与核糖体亚基和单个核糖体分离,因为它们通过蔗糖梯度迁移得更快(Noll,2008).事实上,由于每个核糖体都增加了大量质量,因此可以将包含各种数量核糖体的多聚体彼此分离。制备细菌裂解物并使用蔗糖梯度沉降解析所有核糖体部分是简单的。生成的多聚体\'profile\'可以提供单元格中翻译活动的快照。多聚体分析已用于研究突变和/或生长条件对翻译的影响,并解决特定的细胞成分是否与翻译机制相关(Powers和Noller,1990年;格雷戈里等人。,1994;Moine和Dahlberg,1994;Firpo等人。,1996年;弗雷德里克等人。,2000;Ataide等人。,2009;Melamed等人。,2009;Saini等人。,2009).结合其他技术,多体分析已用于推导某些平移阶段的速率常数(例如,initiation,伸长率,终止)(Arava等人。,2003;2005)。最后,在生化实验中使用纯化的多聚体有助于分离和表征翻译因子,例如核糖体再循环因子(RRF)(Hirashima和Kaji,1972年;藤原等人。,2001年;Hirokawa等人。,2002年;伊藤等人。,2002).这里,我们描述了一种从大肠杆菌中制备和分析多体的简单方便的方法,这通常适用于许多细菌。我们还讨论了影响mRNA核糖体密度的参数,在解释多体轮廓时应该记住这一点。
In actively growing cells, the rate of translation initiation is relatively rapid. As a result, multiple ribosomes simultaneously engaged in translation become spaced along single mRNA molecules. These structures, termed polysomes, can be separated from ribosomal subunits and single ribosomes because they migrate faster through sucrose gradients (Noll, 2008). In fact, polysomes containing various numbers of ribosomes can be resolved from one another since each ribosome adds substantial mass. It is straightforward to prepare bacterial lysates and resolve all the ribosomal fractions using sucrose gradient sedimentation. The resulting polysome \'profile\' can provide a sort of snapshot of the translation activity in the cell. Polysome analysis has been used to study the effects of mutations and/or growth conditions on translation and to address whether particular cellular components are associated with the translational machinery (Powers and Noller, 1990; Gregory et al., 1994; Moine and Dahlberg, 1994; Firpo et al., 1996; Fredrick et al., 2000; Ataide et al., 2009; Melamed et al., 2009; Saini et al., 2009). In combination with other techniques, polysome analysis has been used to deduce rate constants for certain phases of translation (e.g., initiation, elongation, termination) (Arava et al., 2003; 2005). Finally, use of purified polysomes in biochemical experiments has been instrumental for the isolation and characterization of translation factors such as ribosome recycling factor (RRF) (Hirashima and Kaji, 1972; Fujiwara et al., 2001; Hirokawa et al., 2002; Ito et al., 2002). Here, we describe a simple and convenient method of preparing and analyzing polysomes from Escherichia coli, which should be generally applicable to many bacteria. We also discuss parameters that influence the ribosome density on mRNA, which should be kept in mind when polysome profiles are being interpreted.