Mutations of Non-canonical Base-pairs in the 3′ Major Domain of Escherichia coli16 S Ribosomal RNA Affect the Initiation and Elongation of Protein Synthesis

Abstract

Three single-point mutations and two multibase substitutions were introduced into the lower half of the 3′ major domain of Escherichia coli16 S ribosomal RNA. The three single mutations were located in helix 29 (U1341C) or in helix 43 (U1351C or A1357C) and replaced highly conserved non-canonical base-pairs with Watson-Crick base-pairs. The two multibase substitutions were located at the base of helix 42, where they transformed an irregular portion into a Watson-Crick segment. Each of the single mutations could be expressed in vivofrom the rrnBoperon of a multicopy plasmid under control of constitutive promoters, and none of them affected growth-rate. However, mutation A1357C, but not U1341C or U1351C, severely retarded cell growth, when expressed together with another mutation in the upper half of the 3′ major domain, C1192U. The latter mutation is located in helix 34 and abolishes the binding of spectinomycin, a protein synthesis inhibitor. The proportion of mutated ribosomes was high in polysomes, suggesting that the A1357C and C1192U double mutation did not affect the initiation but rather the elongation of protein synthesis. The effect of the double mutation reveals a functional interplay between helices 34 and 43. Furthermore, an interaction between helices 34 and 43 was also suggested by studies of protection by spectinomycin against chemical attack, that showed that the binding site of spectinomycin was restored with ribosomes bearing another double mutation, U1351C and C1192U. In contrast to the single mutations, the multiple mutations in helix 42 could not be expressed in vivounder control of the strong constitutive promoters, but could be expressed under control of the weaker, thermoinducible λP Lpromoter. They did not affect cell growth, whether expressed in the absence or the presence of the C1192U mutation. However, under conditions where protein synthesis depended exclusively on ribosomes with plasmid-encoded rRNA, cells transformed with plasmids altered in helix 42 could not grow. Analysis of the plasmid-borne 16 S rRNA distribution in bacteria transformed with these mutant plasmids showed that mutant 16 S rRNA was present in a high proportion in the free 30 S subunits but was underrepresented in 70 S ribosomes and polysomes. Extension inhibition assays (toeprinting) demonstrated that this altered distribution resulted from an impaired capacity of the mutant 30 S subunits to form translation initiation complexes.