Analysis of polysomes from bacteria

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.