BASE COMPLEMENTARITY IN HELIX-2 OF THE CENTRAL PSEUDOKNOT IN 16S RIBOSOMAL-RNA IS ESSENTIAL FOR RIBOSOME FUNCTIONING

Helix 2 of the central pseudoknot structure in Escherichia coil 16S rR NA is formed by a long-distance interaction between nt 17-19 and 918-9 16, resulting in three base pairs: U-17-A(918), C-18-G(917) and A(19)- U-916. Previous work has shown that disruption of the central base pai r abolishes ribosomal activity, We have mutated the first and last bas e pairs and tested the mutants for their translational activity in viv o, using a specialized ribosome system, Mutations that disrupt Watson- Crick base pairing result in strongly impaired translational activity, An exception is the mutation U-916-->G, creating an A.G pair, which s hows almost no decrease in activity, Mutations that maintain base comp lementarity have little or no impact on translational efficiency, Some of the introduced base pair substitutions substantially alter the sta bility of helix 2, but this does not influence ribosome functioning, n either at 42 nor at 28 degrees C. Therefore, our results do not suppor t models in which the pseudoknot is periodically disrupted, Rather, th e central pseudoknot structure is suggested to function as a permanent structural element necessary for proper organization in the center of the 30S subunit.