Polyphenylalanine synthesis by purified in vitro reconstituted Escherichia coli 30S particles. Reconstitutions were performed under the conditions of Alix and Nierhaus (2003). Polyphenylalanine generated by purified 30S particles reconstituted under normal, high-temperature (42°C) conditions (solid circles) compared to natural 50S subunits alone (open squares), or particles reconstituted at low temperatures (20°, 21S) (solid diamonds), or as for the 21S reconstitution but in the presence of the DnaK chaperone system (solid squares). Chaperone conditions were similar to those of Maki et al. (2002) (16S rRNA:DnaK:DnaJ:GrpE 1:1:1:2 with 1mM ATP). The polyphenylalanine synthesis was carried out essentially as described (Southworth et al. 2002), by incubating 30S particles and 50S subunits (0.3 μM) with poly-uridine (0.35 mg/mL), 14C Phe-tRNAPhe, His-tagged EF-G (0.3 μM), His-tagged EF-Tu (2 μM), GTP (1.4 mM), phosphoenolpyruvate (3.5 mM), and pyruvate kinase (14 μg/mL). Reactions were carried out in 80 mM K+-HEPES pH 7.6, 13 mM MgCl2, and 100 mM KCl.
