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

Abstract

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, but it is integrated efficiently during any of the assembly steps in vitro. The 47S particle, reconstituted in 2 steps and lacking 5S RNA, shows low but significant activity in many functional tests. High activity is 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.