THE CENTRAL PSEUDOKNOT IN 16S RIBOSOMAL-RNA IS NEEDED FOR RIBOSOME STABILITY BUT IS NOT ESSENTIAL FOR 30S INITIATION COMPLEX-FORMATION

To examine the function of the central pseudoknot in 16S rRNA, we have studied Escherichia coil 30S subunits with the A(18) mutation in this structure element, Previously, this mutation, which changes the centr al base pair of helix 2, C-18-G(917) to an A(18)xG(917) mismatch, was shown to inhibit translation in vivo and a defect in initiation was su ggested, Here, we find that the mutant 30S particles are impaired in f orming 70S tight couples and predominantly accumulate as free 30S subu nits, Formation of a 30S initiation complex, as measured by toeprintin g, was almost as efficient for mutant 30S subunits, derived from the t ight couple fraction, as for the wild-type control, However, the A(18) mutation has a profound effect on the overall stability of the subuni t, The mutant ribosomes were inactivated by affinity chromatography an d high salt treatment, due to easy loss of ribosomal proteins, Accordi ngly, the particles could be reactivated by partial in vitro reconstit ution with 30S ribosomal proteins, Mutant 30S subunits from the free s ubunit fraction were already inactive upon isolation, but could also b e reactivated by reconstitution, Apparently, the inactivity in initiat ion of these mutant 30S subunits is, at least in part, also due to the lack of essential ribosomal proteins, We conclude that disruption of helix 2 of the central pseudoknot by itself does not affect the format ion of a 30S initiation complex, We suggest that the in vivo translati onal defect of the mutant ribosomes is caused by their inability to fo rm 70S initiation complexes.