Analysis of the function of E. coli 23S rRNA helix-loop 69 by mutagenesis

We scanned the loop of helix 69 by mutagenesis and analyzed the mutant ribosomes using a plasmid-borne IPTG-inducible expression system. We assayed the effects of 23S rRNA mutations on cell growth, contribution of mutant ribosomes to cellular polysome pools and the ability of mutant ribosomes to function in cell-free translation. Mutations A1912G, and A1919G have very strong growth phenotypes, are inactive during in vitro protein synthesis, and under-represented in the polysomes. Mutation Ψ1917C has a very strong growth phenotype and leads to a general depletion of the cellular polysome pool. Mutation A1916G, having a modest growth phenotype, is apparently defective in the assembly of the 70S ribosome.Mutations A1912G, A1919G, and Ψ1917C of 23S rRNA strongly inhibit translation. Mutation A1916G causes a defect in the 50S subunit or 70S formation. Mutations Ψ1911C, A1913G, C1914A, Ψ1915C, and A1918G lack clear phenotypes.High-to-medium-resolution structures of the ribosome have by their ability to generate structure-based functional hypotheses radically changed the way the ribosome is studied. One of the more intriguing results that has come from structural studies is the extraordinary number of roles attributed to a single 19 nt helix-loop, H69 of 23S rRNA (Fig 1). Crystallographic studies of the Thermus thermophilus ribosome [1] and cryo-EM studies of E. coli ribosomes [2,3] have made it evident that H69 is a component of both the A and P sites with an ability to simultaneously contact two tRNAs. It contacts the D-stem and D-stem junction of the A site tRNA by the loop residues 1913–1915 and the same parts of the P site tRNA by backbone-backbone interactions with stem nucleotides 1908, 1909, 1922 and 1923 [1] (Fig 1). In addition, H69 loop residues 1912, 1913, 1914 and 1918 contact 16S rRNA H44, thus forming the intersubunit bridge B2a [1,2]. Chemical cross-linking and footprinting data further corroborates the close proximity of H69 to intersubunit contact area [4