Role of 5S RNA in assembly and function of the 50S subunit from Escherichia coli

Total reconstitution experiments performed under various conditions revealed that 5S RNA plays an important role during the last assembly step in vitro leading to an active 50S particle. For the preceding steps this RNA species is dispensable. However, 50S RNA can be integrated efficiently during any of the assembly steps in vitro. The 47S particle, reconstituted in two steps and lacking 5S RNA, shows low but significant activity in many functional tests. High activity could be obtained by incubating this particle with 5S RNA alone, demonstrating the importance of the 5S RNA in generating an active ribosomal conformation. In particular, the activity of the peptidyltransferase (peptidyl-tRNA:aminoacyl-tRNA N-peptidyltransferase; EC 2.3.2.12) center is drastically influenced by 5S RNA. No significant factor-dependent tRNA binding to the A-site was observed with the 47S particle, in contrast to the corresponding P-site binding. The elongation factor G dependent GTPase activity was not affected by the lack of 5S RNA.