Kinetic impairment of restrictive streptomycin-resistant ribosomes

Abstract

Comparisons in vivo and in vitro of wild-type and otherwise isogenic bacteria with five different mutant alleles of the gene (rpsL) specifying ribosomal protein S12, all resistant to high levels of streptomycin, show that the streptomycin-resistant (Smr) phenotype can be subdivided into major groups: restrictive and non-restrictive. The restrictive bacteria have a characteristically lower frequency of nonsense suppression in vivo, and are also slower than the wild type in their rate of protein synthesis. Non-restrictive Smr bacteria on the other hand do not differ significantly from the wild type either in nonsense suppression frequencies or in the rate of translation. A complementary pattern is seen in vitro, where ribosomes from the restrictive Smr bacteria translate poly(U) with a significantly lower missense error frequency than wild-type ribosomes, and also show an increased Michaelis constant (K _M) with respect to their substrate, i.e. ternary complexes. Both effects are correlated with the more aggressive proofreading function that is characteristic of these restrictive ribosomes. In contrast, ribosomes isolated from the non-restrictive Smr bacteria do not show any major difference in either proofreading or missense error in vitro when compared to the wild type.