Effects of mutations in the junction between helices 5 and 6 of the 16S rRNA upon 30S biogenesis
Rappe, Mollie C
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The ribosome is a large, compact RNA machine stabilized by conserved sequence motifs. The junction between helices 5 and 6 of the 16S 5’ domain undergoes transient structural rearrangements during in vitro assembly and the sequence of Right Angle motif consensus sequence but not the structure. In this work I will show that in vitro rRNA folding, r-protein binding, and RNP chemical footprinting reveal mild defects such as a greater dependence on Mg2+ when folding in low K+, approximately two-fold weaker r-protein association, and local structural perturbations. In vivo, the 30S ribosomes carrying mutations in this junction cannot support life, as they fail to mature. Neither an increase in growth temperature nor overexpression of ribosome assembly factors improves mutant pre-rRNA processing or polysomal localization. In vivo hydroxyl radical footprinting of wild type and mutant 16S rRNA revealed solvent exposures at helices 35-37 of the 3’ head domain and cleavages at helix 2. This suggests that late r-protein S2 and the native tertiary interactions with other domains of the 30S ribosome are not present in the J5/6 Triple mutant. These data are consistent with a hypothesis that the J5/6 mutants perturb central pseudoknot formation and proper docking of the 3’ head domain, perhaps communicated from the junction to helix 3 through helix 15 packing.