The role of 16S RNA in ribosome function: single base alterations and their effect on in vitro protein synthesis

An in vitro system developed for the site-specific mutagenesis of 16S RNA of Escherichia coli ribosomes (Krzyzosiak et al., Biochemistry 26, 2353-2364, 1987) was used to make 10 single base changes around C1400, the residue known to be at the decoding site. C1400 was replaced by U, A, or G, 5 single base deletions at and to either side of C1400 were made, and C or U was inserted next to C1400. Another mutant possessed 7 additional nucleotides at the 3' end of the 16S RNA such that a stem and loop involving the anti-Shine-Dalgarno sequence could form. Another series of 8 mutants tested hypothetical base-pairing between C1404, G1405 and C1496, G1497. The activity of these 19 mutants in A and P site binding, and in initiation-dependent and initiation-independent peptide synthesis was determined. None of the base substitutions of C1400 were strongly inhibitory. The insertions and deletions completely blocked initiation-dependent peptide synthesis but markedly stimulated the initiation-independent reaction. The effects of tRNA binding were variable. The only alteration to block all ribosomal function was the deletion of G1401. The extra stem and loop at the 3'-end blocked initiation-dependent peptide synthesis, but all other assays were normal. The mutants which break and reform the hypothetical base-pairs had a functional pattern that suggest the contiguous base-pairs do exist and are functionally important.